HISTO LOGIC ® Vol. XXXVII, No. 1, May 2004 Materials and Methods Animal Model The surgically induced medial meniscal tear procedure is performed on 300-400 gram rats. The medial collateral ligament is transected and the medial meniscus is reflected medially toward the femur and then cut. The cut is made through the full thickness to simulate a complete tear. As a result, rapid cartilage degeneration occurs within 3 weeks postsurgery. 1,3 Testing of potential antiarthritis drugs is initiated prior to or after surgery and is continued for 3 weeks with 20 animals per treatment group to account for variability in lesion severity.At necropsy, the right knee joint is trimmed of muscle and connective tissue, and the patella is removed to allow proper fixation of the joint. The femur and tibia are transected with a rongeur some distance from the joint to avoid fragmentation of the bone around the joint area. The joint is then placed in 10% neutral buffered formalin (fixative) and allowed to assume a natural degree of flexion. 1,2 Decalcified Bone Sections in Surgically Induced Osteoarthritis in Rat Amy Rutledge, BS, HTL(ASCP) Elizabeth Chlipala, BS, HTL(ASCP) Premier Histology Laboratory, LLC University of Colorado Boulder, CO lizchlipala@pr emierhistology .com Introduction Osteoarthritis (OA) is a degenerative joint disease that causes the breakdown of the joint’s cartilage. It is one of the oldest and most common types of arthritis currently affecting more than 20 million adults in the United States, and estimated by 2030 to affect 70 million Americans. 1 The surgically induced animal model of osteoarthritis in rats is used to study the pathogenesis of cartilage degeneration and evaluate potential antiarthritis drugs for clinical use. 2 By creating a medial meniscus tear surgically, one can study the kind of rapid and severe cartilage degeneration that occurs in some stages of human osteoarthritis. 1 These morphological changes can only be demonstrated through the histological preparation of the knee joint. Histological evaluation of the decalcified, paraffin-embedded toluidine blue-stained sections is performed to determine the severity of cartilage degeneration, the presence of osteophyte formation, and the efficacy of a potential drug. Managing Editor, Gilles Lefebvre Scientific Editor, Vinnie Della Speranza, MS, HTL (ASCP) HT, MT 1 IN THIS ISSUE Decalcified Bone Sections in Surgically Induced Osteoarthritis in Rat …………… 1 Preparation and Snap Freezing of Murine Tissues for Research Immunohistochemistry and Routine Hematoxylin & Eosin Staining …………………………… 4 A Novel Technique to Embed Tissues for Frozen Section Cryotomy …………… 7 Debunking an Urban Legend in Bone and Cartilage Histotechnique: Safranin O Fast Green Will Stain Proteoglycans After EDTA Decalcification …………………… 10 Immunohistochemical Staining Techniques to Demonstrate Treponema pallidum Spirochetes ……………………………… 14 Fig. 1. Photomicrograph of knee joint that has been assessed histologically for cartilage degeneration and osteophyte formation. The red lines divide the tibial plateau into 3 zones: Z1, Z2, and Z3. A micrometer measurement is taken across areas of degeneration; the purple line denotes the significant matrix loss. The osteophyte formation measurement is denoted by the yellow line.
Transcript
HISTOLOGIC ®
Vol. XXXVII, No. 1, May 2004
Materials and MethodsAnimal ModelThe surgically induced medialmeniscal tear procedure isperformed on 300-400 gram rats.The medial collateral ligament istransected and the medial meniscusis reflected medially toward thefemur and then cut. The cut is madethrough the full thickness tosimulate a complete tear. As aresult, rapid cartilage degenerationoccurs within 3 weeks postsurgery.1,3
Testing of potential antiarthritisdrugs is initiated prior to or aftersurgery and is continued for 3 weekswith 20 animals per treatmentgroup to account for variability inlesion severity. At necropsy, theright knee joint is trimmed ofmuscle and connective tissue, andthe patella is removed to allowproper fixation of the joint. Thefemur and tibia are transected witha rongeur some distance from thejoint to avoid fragmentation of thebone around the joint area. Thejoint is then placed in 10% neutralbuffered formalin (fixative) andallowed to assume a natural degreeof flexion.1,2
Decalcified Bone Sections in SurgicallyInduced Osteoarthritis in Rat
IntroductionOsteoarthritis (OA) is a degenerative joint disease that causes thebreakdown of the joint’s cartilage. It is one of the oldest and mostcommon types of arthritis currently affecting more than 20 million adultsin the United States, and estimated by 2030 to affect 70 millionAmericans.1 The surgically induced animal model of osteoarthritis in ratsis used to study the pathogenesis of cartilage degeneration and evaluatepotential antiarthritis drugs for clinical use.2 By creating a medialmeniscus tear surgically, one can study the kind of rapid and severecartilage degeneration that occurs in some stages of humanosteoarthritis.1 These morphological changes can only be demonstratedthrough the histological preparation of the knee joint. Histologicalevaluation of the decalcified, paraffin-embedded toluidine blue-stainedsections is performed to determine the severity of cartilage degeneration,the presence of osteophyte formation, and the efficacy of a potential drug.
Managing Editor, Gilles LefebvreScientific Editor, Vinnie Della Speranza,
MS, HTL (ASCP) HT, MT
1
IN THIS ISSUE
Decalcified Bone Sections in SurgicallyInduced Osteoarthritis in Rat …………… 1
Preparation and Snap Freezing of MurineTissues for Research Immunohistochemistryand Routine Hematoxylin & Eosin Staining …………………………… 4
A Novel Technique to Embed Tissuesfor Frozen Section Cryotomy …………… 7
Debunking an Urban Legend in Bone andCartilage Histotechnique: Safranin O FastGreen Will Stain Proteoglycans AfterEDTA Decalcification …………………… 10
Fig. 1. Photomicrograph of knee joint that has been assessed histologically for cartilage degeneration andosteophyte formation. The red lines divide the tibial plateau into 3 zones: Z1, Z2, and Z3. A micrometermeasurement is taken across areas of degeneration; the purple line denotes the significant matrix loss.The osteophyte formation measurement is denoted by the yellow line.
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HistologyThe knee joints are fixed in 10%neutral buffered formalin for48 hours and then placed into 5%formic acid for 3-6 days to decalcify.Once decalcified, the knee joints arecut into approximately 2 equalhalves in the frontal plane, using thecollateral ligament as a landmark.
The joints are processed for paraffinembedding and sectioned at 8 µmfor toluidine blue staining. An initialsection is cut followed by two stepcuts at 200 µm intervals, yielding atotal of 3 sections per knee.
ResultsThe knee joints are evaluated forseverity of the medial femoral andmedial tibial cartilage degeneration,and also for osteophyte formation.The primary area that is evaluatedfor cartilage damage is the medialtibia. The medial tibia is divided into3 zones (inner, middle, outer), andeach zone is scored separately. Thefollowing system is used to score thecartilage degeneration in each zone.
Initially, the depth of chondrocyteand proteoglycan loss withfibrillation is calculated by usingthe following criteria:
1=minimal; superficial zone only
2=mild; extends into the uppermiddle zone
3=moderate; well into the middlezone
4=marked; into the deep zonebut not to tidemark
5=severe; full thicknessdegeneration to tidemark
Next, the area of cartilagedegeneration involved is assessed as1/3, 2/3, or 3/3 of the surface of thesection. The score is then multipliedby 1, 2, or 3 to reflect the extent ofthe tibial plateau that is involved.
For tibial degenerative change, amicrometer measurement is takenacross the portion of medial tibiathat demonstrates any cartilagedegeneration. Then a second
Fig. 2B. Photomicrograph of medial aspect of knee joint from rat which had unilateral medial meniscal tear surgery3 weeks previously. Focal severe cartilage degeneration, characterized by chondrocyte and proteoglycan loss as wellas fibrillation, is present on the medial tibial plateau. Toluidine blue. Original magnification 50X
Fig. 2A. Photomicrograph of medial aspect of a normal rat knee joint. There is no chondrocyte or proteoglycan loss.Toluidine blue. Original magnification 50X
Fig. 3. Photomicrograph of the medial aspect of a knee joint from rat, which had medial meniscal tear surgery.Toluidine blue. Original magnification 50X
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Total Tibial Degeneration Width
VehicleTreatment
Mea
n�
SE
Tib
ial C
arti
lage
Deg
ener
atio
n (�
m)
Compound X
n=20/groupPercent on bars is percent inhibition as compared to vehicle.
1500
1000
500
0
15.4%
13%
Significant Tibial Degeneration Width
Zone 1 (Outer)
VehicleTreatment
Mea
n –
SE T
ibia
l Car
tila
ge D
egen
erat
ion
Scor
e(S
core
0-5
, mea
n of
3 s
ecti
ons)
Compound X
n=20/groupPercent on bars is percent inhibition as compared to vehicle.
8
7
6
5
4
3
2
1
0
12%
19%
50%
17%
Zone 2 (Middle)Zone 3 (Inner)Total Medial Tibia
Fig. 4A. Cartilage degeneration score of sample vehicle and compound data. Graphical representation of the major histopathological parameters includingthe mean, standard error, statistical analysis, and percent inhibition.
Fig. 4B. Cartilage degeneration width of sample vehicle and compound data.
measurement is taken of the areaof degeneration that has resulted insignificant matrix loss (greater than50% of the cartilage thickness) in aneffort to further quantitate the moreserious changes.
Osteophytes are measured andcategorized into small, medium, andlarge using an ocular micrometer.
Osteophyte evaluation isdetermined by the following criteria:
1=small, up to 299 µm
2=moderate, 300-399 µm
3=large, �400 µm
ConclusionsThe information acquired from thehistopathology allows the evaluationof potential antiarthritis drugs.The results acquired from thisanimal model of disease show theeffects of compounds on proteo-glycan degradation and osteophyteformation. In addition, activity ofmetalloproteinase inhibitors andother antiarthritis compounds maybe detected. This rat model mimicshuman traumatic osteoarthritisand demonstrates morphologicalchanges of pathogenesis thatcompare to human disease.
Since the data generated from thehistology cannot be captured byany other means, it is importantthat each specimen is handledappropriately. Great care must betaken to assure that the knee jointis bisected into two equal halves,and processed and embeddedproperly so that the subsequentlevels demonstrate lesion severitywith consistency.
References1. Bendele AM, McComb J, Gould T, et al. Animal
models of arthritis: relevance to human disease.Toxicol Pathol. 1999;27:134-142.
2. Clarke KA, Heitmeyer SA, Smith AG, Taiwo YO.Gait analysis in a rat model of osteoarthrosis. PhysiolBehav. 1997;62:951-954.
3. Janusz MJ, Bendele AM, Brown KK, Taiwo YO,Hsieh L, Heitmeyer SA. Induction of osteoarthritis inthe rat by surgical tear of the meniscus: inhibition ofjoint damage by a matrix metalloproteinase inhibitor.Osteoarthritis Cartilage. 2002;10:785-791. Errata in:Osteoarthritis Cartilage. 2002;10:905. OsteoarthritisCartilage. 2003;11:299.
Preparation and SnapFreezing of Murine
Tissues for ResearchImmunohistochemistry
and RoutineHematoxylin & Eosin
StainingGayle Callis, MT, HTL(ASCP)HT
Veterinary Molecular BiologyMontana State University
Murine tissues can presenthistotechnicians with multiplecryomicrotomy problems rangingfrom the initial tissue collection tothe final frozen section. Formalin-fixed, paraffin-embedded (FFPE)tissues are unusable whenimmunohistochemical (IHC)staining for rat anti-mouse-CD4
and -CD8 lymphocytes is needed.Antigen unmasking and enzymedigestion are unsuccessful atrecovering these antigens. Thesemurine CD markers are notoriousfor always giving negative resultsafter FFPE tissue preparation.Because our laboratory routinelystains for CD4 and CD8 along with apanel of other CD markers on serialsections from a single tissue sample,we have abandoned FFPE tissues infavor of cryomicrotomy methods.This article will discuss tissuecollection, snap freezing, and frozenblock storage methods usedsuccessfully in our laboratory.
Tissue CollectionAnimal euthanasia is performedaccording to strict protocols setforth and monitored by ouruniversity animal care committees.After euthanasia, tissues aredissected out quickly and snapfrozen within minutes to preventdamaging autolysis and potentialantigen diffusion. Most tissues areremoved without problems butsome organs must be handled inspecial ways. Prior to embedding,the plastic Tissue-Tek® Cryomold®
(Sakura Finetek, Torrance, CA),
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Fig. 6A. After several days in decal, the ends of the boneare trimmed and the knee joint is placed in forcepsalong the patellar groove to hold the orientation whilecutting in half. The collateral ligament is used as alandmark to achieve two equal halves, which is a criticalstep in the grossing.
Fig. 5. Gross photograph of rat knee demonstratingnormal flexion. The majority of the connective tissueand muscle have been removed along with the patellato allow penetration of the fixative into the joint.
Fig. 6B. Demonstration of what is achieved when usingthe collateral ligament as a guide to cut the knee jointinto two equal halves.
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designed to support tissue cassettes,is trimmed to remove the 3 plasticedges; it now resembles the roundTissue-Tek® Cryo3
® biopsy moldwith extended tab. This tab is heldwith forceps during snap freezingand the trimmed mold is easier toinsert into a 50 ml tube for freezerstorage. Smaller tissues such aslymph node, spleen, liver slices, andbrain (either whole or bisected atmidline) are embedded separately.Different tissues, such as spleen andintestine, have different sectioningqualities and are never put togetherin the same block. The followingtissues require special handling inorder to cryosection properly.
Whole lung is filled with OCT®
(Sakura Finetek, Torrance, CA) byexposing the trachea and makinga tiny V-shaped cut on top of thetrachea with care—do not cutacross this tubular structure. Thesevered trachea retracts into thechest area and cannot be retrievedeasily for filling the lung with OCT.A dulled 18 gauge hypodermicneedle attached to a 3 ml syringe isinserted into this V cut and pushedgently toward the lung withoutpuncturing the trachea wall.
Approximately 2.5 ml OCT isinjected slowly into the lung andobserved carefully to avoidoverfilling lung air spaces. Thepinkish-white deflated lung expandsinto a translucent, inflated lung.Delicate alveoli are severelydamaged by overfilling the lungwith OCT. To prevent OCT leakage,a mosquito hemostat forceps is usedto clamp off the trachea as theneedle is pulled out. The lung isremoved from the pleural cavityand separated from the heart. Thelung and heart are embedded inOCT separately in appropriatelysized Cryomolds.
Brain can be embedded whole,bisected midsagitally, or cut intotransverse/cross-sections from frontto back of the brain.
Spinal cord is removed by severingthe vertebral column at the base of
the skull near the last lumbarvertebra. The cord is forced out ofthe vertebral column by injectingphosphate buffered saline (PBS)into the lumbar vertebral openingso the cord ejects out of the widercervical vertebral opening. The cordis cut into shorter 3 mm lengths andembedded in a small amount ofOCT to maintain a flat, side-by-sideorientation of these pieces. Prior tosectioning, cord blocks are turnedon end and surrounded by extraOCT on the block holder to obtaintransverse (cross) sections of thespinal cord.
Small intestine (whole) istransected below the stomach andabove the cecum and dissected outcarefully to avoid puncturing theintestine. The entire intestine isrinsed with PBS to remove fecalmatter, followed by injection ofOCT into the lumen using a dull16 gauge needle on a 10 ml syringe.Avoid air bubbles in the syringeand inside the gut lumen. We preferthe intestine distended with OCTfor midsagittal sectioning, whichreveals Peyer’s patches (gut-associated lymphoid tissue, orGALT) at both the outer (serosal)and inner (lumenal) surfaces withdistinct intestinal villi. Theduodenum is distinguished by atiny constriction of small intestinejust before the jejunum.Approximately 5 to 6 lengths ofintestine are cut and each length isoriented into a concentric circle inan empty, large Cryomold (Fig. 1).Each length is labeled as to
approximate location along thesmall intestine, eg, duodenum (D),jejunum (J1, J2), and ileum (I1, I2).It is important to note that needlegauge (size) will vary according tothe age of the animal, with youngeranimals needing a smaller gauge(and conversely, a larger gaugeneeded for older animals), in orderto have a tight fit into either theintestine or trachea lumen. Usingthe correct needle gauge permitseasy insertion and prevents tearingof tissue and leaking of OCT orPBS during injection procedures.
Calcified bone can either beembedded directly into OCT orcoated with 4% polyvinyl alcohol(PVA, water soluble, 35,000 to70,000 MW, Sigma), then snapfrozen without further embedmentin OCT. We successfully substituted4% PVA with OCT diluted 1:1 withPBS. A dry ice/hexane immersionfreezing method is used for bone.1,2
Bone can shatter if frozen directlyin liquid nitrogen (LiqN2) or2-methyl butane cooled by LiqN2.Mouse nasal turbinates are toolarge for Cryomolds so they areembedded with the nose endoriented on the bottom of thedeeper 22 x 40 mm Peel-A-Way®
disposable molds (Polysciences,Warrington, PA). This results in alonger, potentially unstable blockextending away from a metal chuckduring cryosectioning. Bone blocksare mounted onto block holderswith 2% methyl cellulose (Aldrich)for a firmer hold that helps preventvibration during sectioning.
There is a caveat, however; not allcryomedia work well with ourmurine tissue applications. Wediscovered by trial and error thatone cryomedium failed to holddistended intestine properly duringcryosectioning and resulted incompressed gut frozen sectionswith distorted, torn villi. Anothercryomedium always pulled awayfrom spleen and other tissuesduring sectioning, yielding curled,folded tissue sections. For somereason, these cryomedia failed tointerface with our mouse tissues
Fig. 1. Concentric circle orientation of small intestinewith Peyer’s patches (red) located on the outerserosal surface.
during snap freezing, sectioning,or both. It is recommended thatlaboratories test several cryomediato optimize their particularcryosectioning needs. Failure of acryomedium for murine cryotomydoes not mean that the medium isdefective; it is more likely that thecryomedium was never testedinitially for murine researchapplications, but only for humantissue application.
Snap Freezing MethodsOur laboratory uses three snapfreezing methods to produce asmany as 30 or more blocks duringa day’s freezing session. We nolonger use LiqN2-cooled 2-methylbutane because it is too time-consuming to recool the solventbetween each block. Ultracold(-80ºC) freezers and cryostats withPeltier or heat extractor devices arenever used to snap freeze tissuesfor research purposes. The tissuefreezes too slowly with thesedevices and tissue morphology isdamaged due to water ice crystalformation, or freezing artifact.Although we have not repairedfreezing artifact on murine tissues,one may want to try the recentlypublished method for frozen tissue
recovery.3 Sucrose (20%-30%)cryoprotection is not used to reducewater ice crystal formation with ourfresh, unfixed tissues, but it is usedwith paraformaldehyde-fixedtissues destined for snap freezing.
We prefer snap freezing tissuesby immersion in a dry ice/2-methylbutane mixture at approximately-78°C (Fig. 3). Hexane can besubstituted for 2-methyl butanefor any tissue and is commonly usedto snap freeze calcified bone.1Immersion methods permit longfreezing sessions, producing as manyblocks as necessary withoutrecooling the solvent. Anothermethod that eliminates volatile,toxic solvents is a Petri dish floatingin LiqN2 (Fig. 4) with the dishsupported to avoid tipping over intothe LiqN2. We use either a large,solid aluminum block or a metaltube rack placed inside a styrofoambox with enough LiqN2 added to thebox so that the dish “canoes” on thesurface of this ultracold liquid. ThePetri dish separates the Cryomoldsfrom direct contact with the LiqN2 ,which must be kept out of the dish toprevent cracked OCT and shatteredtissues. Rarely, a solid dry ice blockis used to snap freeze very tiny lymphnodes in small molds in order to havea totally flat block face (Fig. 2), eventhough the Petri dish/LiqN2 methodresults in perfectly flat blocks. Thedry ice block method4 freezes tissueslower than either the immersion or
LiqN2/Petri dish method and willproduce freezing artifact in largertissues such as spleen. 2-Methylbutane and hexanes are majorhealth, fire, and explosion hazardsand must be stored in explosion-proof freezers.5 Liquid nitrogenshould be used in a well-ventilatedroom to avoid suffocating nitrogenfumes and also to prevent severefrostbite.
Dry Ice Block Method4
A Cryomold with OCT-embeddedtissue is used on a solid dry iceblock (-78ºC). Dry ice should bereflattened on a warm surface toavoid reusing the depression left bya previously frozen block. This willprevent a slowdown in freezing dueto air space between the mold andthe dry ice depression (see Fig. 2).
Dry Ice/2-Methyl Butane(or Hexane) MixtureImmersion MethodA styrofoam box containingcrushed dry ice surroundinga 2-methyl butane-filled beakerprecools the solvent 30 minutesbefore adding dry ice directly intothe solvent. Do not add dry icedirectly into room temperature2-methyl butane or violentbubbling with spillage over beakerrim will create toxic solvent fumes.Add enough dry ice to fill near thetop edge of the beaker for easyrecovery of frozen blocks. Tosnap freeze, the Cryomold with
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Fig. 2. A solid block of dry ice may be used to freeze tinysamples in molds containing OCT in order to obtain atotally flat block face.
Fig. 3. Tissues may be frozen by immersing them into a beaker containing a mixture of dry ice/2-methyl butane.The beaker is surrounded by crushed dry ice in a styrofoam container.
OCT-embedded tissue in Cryomoldbefore snap freezing.
Snap frozen block in dry ice solventmixture.
Styrofoam box with crushed dry icesurrounding a beaker w/ 2-methylbutane.
Beaker w/ 2-methyl butane and dry ice.
Fig. 4. Plastic Petri dish (blue) is supported bya platform to prevent tipping into the LiqN2 insidea styrofoam box.
OCT-embedded tissue is loweredbottom first into the mixture untilthe OCT begins to freeze, then it’sallowed to sink onto the dry icelayer inside the beaker. After theblock is frozen, remove and let it siton dry ice or inside the cryostatchamber to evaporate the explosivesolvent fumes before freezer storage(see Fig. 3).
Petri Dish Floating in Liquid Nitrogen MethodA metal platform surrounded byliquid nitrogen in a styrofoam boxmay be used to freeze OCT-embedded samples in a Petri dish.The platform transfers the cold tothe sample while preventing thePetri dish from tipping into theliquid nitrogen. This method allowsfor better control of the freezingprocess since the sample can beseen throughout the freezingprocess, thus preventing the risk ofoverfreezing and cracking theblock, which can happen withimmersion methods if immersionis prolonged (see Fig. 4).
Frozen Block StorageOur laboratory stores frozen blocksin an ultracold (-80ºC to -86ºC)freezer to prevent both antigenicityloss and freeze-drying of tissues.Sectioned blocks are resealed witha thin OCT layer, wrapped inaluminum foil, and returned toultracold storage. Uncut blocks intrimmed Cryomolds are insertedinto labeled 50 ml screw topcentrifuge tubes and placed instyrofoam tube racks in the freezer(-80ºC). We are fortunate that uncutand/or resealed blocks sectionedafter 6 years of storage at -80°C stillgive excellent immunostainingresults. Length of time for frozenblock storage and successfulimmunostaining can be dependenton a given antigen’s stability towithstand freezer storageconditions from one laboratory toanother. Ultracold freezers areexpensive and indefinite blockstorage presents a problem due tolimited space. Low temperaturefreezers (-27°C to -40°C) are suitablefor short-term (weeks or months)
storage. Automatic self-defrostingfreezers or cryostat chambers arenever used since freeze/thaw cyclescan damage antigens and createfreezing artifact. Unembeddedfrozen tissues can be wrappedtightly in aluminum foil and storedat -80°C until cryosectioning.
Overall, the dissection and snapfreezing methods work well in ourresearch laboratory. It is importantto freeze murine and other animalspecies tissue rapidly forimmunohistochemistry stainingto avoid morphology-damagingfreezing artifact and to stabilizeantigens. Our snap freezingmethods allow us to produce alarge number of tissue blocksper day. Not all laboratories haveultracold freezers but they canpurchase inexpensive, under-the-counter minifreezers withoutautomatic defrost cycles to avoidusing the auto-defrost freezersfound inside many refrigerators.
This article provides someguidelines for initial preparationof tissues for cryomicrotomy.Histotechnology textbooks haveother methods and discussion oncryomicrotomy.6 Information onmouse dissection with excellentphotographs is available atwww.eulep.org/Necropsyof_the_Mouse/index.php orhttp://icg.cpmc.columbia.edu/cattoretti/Protocol/MousePathology/mainPageMousePath2.html. A helpfuland colorful mouse anatomy wallchart is available from theAmerican Association forLaboratory Animal Science.
Bitensky L. Selective depression of metabolic activitiesin cortical osteoblasts at the site of femoral neckfracture. Bone. 1990;11(3):157-161.
2. Tarpley J. Preparation and sectioning of undecalcifiedfrozen rodent long bones and joints using a tapetransfer system. J Histotechnol. 2003;26(1):41-56.
3. Fail R, Della Speranza V. A method to repair freezeartifact in skeletal muscle biopsies. Histologic.2003;36(1):10-11.
4. Callis GM, Jutila M, Kurk S. Snap freezing tissue usingdry ice and cryomolds. Histologic. 1991;21(3):253.
5. Skinner RA, Gaddy D. More than just a mandatoryexercise. Histologic. 2003;36(1):1-4.
6. Gamble M, Bancroft J. Theory and Practice ofHistological Techniques. 5th ed. WB Saunders; 2001.
AbstractSpecimen orientation duringembedding is key to achievingfrozen sections of diagnostic quality.A specimen that has been orientedincorrectly may be irreversiblycompromised if the error has notbeen discovered prior to sectioning.The first section cut may haveruined the entire specimen, such asthe cutting away of mucosal surface.A method used in our laboratorycan facilitate proper specimenorientation when embedding Mohsmicrographic surgery samples forfrozen section, and may also beapplied to virtually any tissuesrequiring frozen section cryotomy.
IntroductionMohs micrographic surgery is aspecialized, highly effectivetechnique for removing skincancers. It was developed in the1930s by Dr. Fredrick Mohs. Mohssurgery differs from other skincancer treatments in that it permitsthe immediate and completemicroscopic examination of theremoved cancer tissue, ensuring thatall roots and extensions of thecancer are eliminated.1 Mohssurgery is commonly used in thetreatment of skin cancers that recurafter previous treatments have beenused, and to preserve normal skin incosmetic areas.
Laboratories employ a numberof different strategies whenembedding tissues for frozensection. In Mohs micrographicsurgery, embedding is criticalbecause the surgical margins mustbe checked and cleared before a
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Fig. 1. A specimen arriving in the laboratory is compared to the surgeon’s mapto confirm correct orientation.
Fig. 2. The tissue is hydrated to prevent drying and to remove excess marking dyein order to make the epidermis more visible.
Fig. 3. The specimen is placed onto a glass slide, deep margin down. Relaxing cutsmay be made to ensure that all margins will be in the same plane for sectioning.
Fig. 4. Tissue must be checked to ensure that no air pockets exist under the tissuebefore freezing can be done.
Fig. 5A. Tissue is placed onto the cryostat freeze bar and lightly coated with OCTwhile a chuck is prepared. In this photo, the knife holder was used to facilitatephotography only, and is not intended to be used in this technique.
Fig. 5B. A chuck is inverted onto the tissue sample as it begins to freeze.
surgeon can declare a patient to betumor free. This is best achieved byhaving the epidermal and deepmargins oriented in the same planewithin the frozen block. Oursurgeons employ different coloredtattoo inks to label the margins,which aids the lab staff in orientingthe specimen.
MethodSamples for frozen section areoriented onto a glass microscopeslide prior to embedding in OCT�
(Sakura Finetek,USA,Torrance,CA),in the same way that a mold is usedto embed tissues in paraffin whenpermanent sections are beingperformed. This method isillustrated in a series of photos toaid the reader in understandinghow specimen orientation ismaintained and frozen sectionembedding is achieved.
The specimen to be examined isdivided, if needed, and markedwith tattoo ink. Lines are drawnon the skin to show the source ofthese specimens and a map of thesurgical site is drawn. The tissueand map are brought to thelaboratory, and the technologistis now ready to embed.
Step 1. The histotechnologistcarefully examines the resectedbiopsy that was obtained from theMohs surgical proceedure. Thesample is then compared to themap provided by the surgeon to
ensure accurate orientation. Theorientation, location, and patientinformation are reconfirmed beforeproceeding. To minimize thenumber of glass slides to beprepared, the block of tissue is keptas large as possible; however, if thetechnologist feels that the tissuespecimen is too large to embedprecisely, it can be subdivided toobtain smaller specimens (see Fig. 1).
Step 2. The tissue is hydrated withwater or saline to prevent dryingand to remove excess marking dyeso that the epidermis is visible. It isimportant to dry off the excesswater before the tissue is embeddedto prevent freeze artifact frominterfering with histologicinterpretation. Once the excess dyehas been removed, the embeddingprocess begins (see Fig. 2).
Step 3. The specimen is placedonto a glass slide, deep margindown, and a chuck is labeled withthe patient’s name andcorresponding accession number.The layer (sample) is carefullyexamined to ensure that the entireepidermal and deep margins arecompletely adhered to the glassslide. It is of utmost importancethat the epidermis is teased downon the slide and the deep tissue isflattened and positioned into thesame plane to yield a true andcomplete surgical margin uponmicroscopic examination. If thetissue is too thick, scoring and
relaxing cuts may be made in thespecimen with a scalpel blade toensure that the epidermis is teaseddown on the glass slide, along withthe deep tissue (see Fig. 3).
Step 4. It is also important to checkfor air pockets that may formbetween the tissue and the glassslide. If air pockets are present,press firmly on the superior aspectof the tissue to expel the entrappedair, thus making contact betweenthe tissue and the underlying glassslide (see Fig. 4).
Step 5. Next, the glass slidecontaining the inked specimen isplaced onto the cryostat freeze barand coated with a thin layer ofOCT, which must be placed over allexposed surfaces and outer edgesof the specimen. Avoid using excessOCT, which results in an enlargedfrozen block face. A block that istoo large will make sectioningmore difficult and can result insection chatter. The OCT-coveredspecimen will turn from a clear toan opaque color. While thistransformation is taking place, putthe labeled chuck on the freeze barand apply OCT. When the chuckand OCT-covered glass slide firstappear opaque, flip the chuck ontothe specimen-covered glass slide.Allow the specimen to freezetogether with the cryostat ata temperature ranging from-28oC to -30oC (see Fig. 5A, 5B).
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Fig. 6A and Fig. 6B. The thumb is used to warm the slide to separate the glass from the frozen block.
Step 6. Warmth from thehistotechnologist’s gloved thumbon the glass will allow the slide tobe removed, leaving a smooth andlevel block face (see Fig. 6A, 6B).One should be able to see theentire tissue sample, which includesthe epidermal edge, deep tissue,and inked margin. The chuck is nowsubmerged into liquid nitrogen forapproximately 3 seconds. There’sno need for any additional OCTon the block face. The block is nowready for sectioning.
DiscussionThere are several advantages to theglass slide embedding techniquecompared to the cryomoldtechnique or the direct method(direct freezing onto the chuck onthe freeze bar while flattening witha heat extractor), techniques thatare often employed in manylaboratories. The glass slidetechnique provides a block facethat is immediately ready forsectioning without the need fortrimming. Furthermore, therequirement that the epidermaland deep margins remain in thesame plane makes it essential thatthe specimen orientation does notchange during freezing—thiscannot be ensured when the directmethod is employed. When usingglass slides, all manipulations areperformed before any OCT isapplied.
Because the tissue block face isn’tcovered with OCT in the glass slideembedding technique, thetechnologist is able to see theepidermal and deep margins before
sectioning begins. This decreasesthe likelihood of tissue being cutaway, affording a chance to melt itdown and re-embed if necessary,before sectioning begins. Withother methods, including the directfreeze bar technique, the block faceis obscured with excess OCT andthe specimen is not visible until theblock has been trimmed, whichrisks critical areas of the tissuebeing cut away.
The glass slide embeddingtechnique is rapid and takes lesstime for the technologist to embedthe specimen than in other freezingmethods. Most important of all, itallows the surgeon to performMohs surgery on more complex,delicate, thin tissues, such as eyelids, due to the accuracy of theembedding and the conservativeway the block is trimmed.Epidermis is seen immediatelyupon embedding, instead of a blockface obscured by OCT.
Finally, the glass slide embeddingtechnique is not only applicableto samples derived from Mohsmicrographic surgery but may alsobe utilized for any tissues requiringfrozen sectioning, including tinyneedle biopsies. This helps avoidunnecessary tissue loss that canotherwise occur during the blocktrimming required with otherfreezing methods.
In today’s electronic worldinformation travels fast. All kindsof information. Good, bad, true,false—it all goes. Urban legends getpassed from coast to coast with theclick of a mouse. Perhaps you arefamiliar with some of these urbanlegends:
• Companies putting addictivesinto soft drinks to create a legionof addicted people who willcontinue buying their product
• Waking up after a night ofpartying in a bathtub of ice withno kidneys because they werestolen and are now on their wayto the highest bidder
• You can’t do a Safranin O FastGreen (SOFG) stain on EDTAdecalcified bone sections
Some of you probably read thatlast one and thought, “That isn’turban legend. EDTA destroysproteoglycans and SOFG is aproteoglycan stain, so of courseyou can’t do this stain on EDTAdecalcified material. After all,this information was posted onHistonet.” However, this commonlyheld belief is simply incorrect.
I have done hundreds of SOFGstain runs since the 1980s. In all
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Fig. 7. The result is perfect sections representative of the entire sample containing all margins.
11
that time I cannot recall everhaving had a procedure fail dueto any specific decalcifying agent.I have worked with bone andcartilage throughout my career andam a frequent lecturer on this topicat the NSH symposium. In recentyears I have received an increasingnumber of questions specificallypertaining to Safranin O Fast Greenstaining and EDTA.
I work in an orthopaedic researchsetting and am quite familiar withthe popularity of EDTA as adecalcifying agent. In particular,one will achieve superior resultswhen performing the TartrateResistant Acid Phosphatase(TRAP) reaction if this procedureis performed on EDTA decalcifiedsections rather than formic aciddecalcified material. Manyorthopaedic researchers also prefersections from EDTA decalcifiedmaterial for histomorphometricand image analysis. Since manyorthopaedic histologic studiesinclude both requirements, there isa tendency to hedge one’s bet and
take the additional time and effortto decalcify via EDTA. Many in theclinical arena do not have the needfor such specificity, but rather havea “need for speed,” which isprovided by decalcifiers whoseprimary constituent componentsare either formic acid orhydrochloric acid. Buffered ornonbuffered EDTA is typicallyregarded as being too slow to meetthe turnaround time demands ofthe clinical laboratory.
All is well and good until someresearcher wants the SOFG onEDTA decalcified material. That’susually when I get “the call” or“the e-mail.”
This time the call came about twoweeks after my bone processingworkshop at the NSH symposium.An investigator at another facilityhad requested SOFG stainssubsequent to the EDTAdecalcification of harvested mouseknees. The technologist thereremembered hearing somethingabout EDTA and SOFG and
decided to call me to see if therewas some way she could salvageher project.
My initial “knee-jerk” reactionwas that there was no cause forconcern. A recent report byKalscheur1 emphasized the value ofSOFG stains in cartilage studies.However, I decided to examine theissue more closely by stainingleftover tissues I had that had beendecalcified in parallel in bothEDTA and formic acid to see what,if any, staining differences might beobserved. In theory, if EDTAdestroys proteoglycans, then onemight expect the EDTA decalcifiedslides to be faintly stained, if at all.
To avoid bias, all of the mouseslides in my study were stainedin the same stain run with ourstandard control. I included tissuefrom different species to evaluatespecies variability. The slides wereexamined by Dr. Larry Suva, ourlab director, in a blind review toavoid observer bias.
Fig. 1. SOFG on 5-micron section of 5% formic aciddecalcified mouse knee. Proteoglycan-containingmaterial stains red. Growth plate stains a darker shadethan articular cartilage. Chondrocytes are identifiable.20X
Fig. 2. SOFG on 5-micron section of 10% EDTAdecalcified mouse knee. Proteoglycan-containingmaterial stains red. Growth plate stains a darker shadethan articular cartilage as in the formic acid decalcifiedprep, although arguably a different shade.Chondrocytes are equally as identifiable in eitherprep. 20X
Fig. 3. SOFG on 5-micron section of 10% EDTAdecalcified rat knee growth plate cartilage. Distinctiveproteoglycan staining is present as denoted by the redareas. 20X
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The evaluated samples includedseveral mouse knees and rat kneesexposed to various decalcificationprotocols (EDTA decalcified forapproximately 3 weeks, endpointdetermined by x-ray, anotherdecalcified for 6 weeks using a14% EDTA solution pH 7.4 anddecalcified in 5% formic acid),as well as rabbit wrist and humanfemoral head, both decalcified in5% formic acid.
In this examination Dr. Suva wasable to identify and countchondrocytes with ease from amongthe various preps; however, henoted that the colors in the formicacid decalcified samples were muchnicer (Fig. 6). We concluded thatthere was a noticeable tinctoraldifference between species, butlittle difference between EDTAand formic acid decalcificationwithin the same species for thepurpose of chondrocyteidentification. A review of theKalscheur images likewise revealedsimilar staining variability.
Dr. Ralph Sanderson, an expertin Syndecan and proteoglycan
research at the University ofArkansas for Medical Sciences,is unequivocal that EDTA doesnot destroy proteoglycans. Hepointed out in a personalcommunication (2003) that EDTAis actually used to harvest cells inculture that are subsequentlystained for proteoglycan, andwould not be used if it had sucha damaging effect.
The caller whose query led to ourstudy of this stain providedelectronic images of the SOFGstains she conducted in herlaboratory. Her staining patternswere consistent with resultsobtained in our lab, but the colorcontrast was far more brilliant thanours, which I believe is due tominor differences in our respectivestain protocols. She also did somedigging and tracked down amessage posted on the Histonetlistserv that could be the genesis ofall this confusion.
The message on Histonet was areply by Gayle Callis to an inquiryregarding decalcification and poorSOFG staining. In her posting,
Callis stated, “EDTA can extractproteoglycans and this extractionmay result in altered tinctoralstaining.” Perhaps some haveinterpreted those remarks tosuggest that SOFG staining couldnot be carried out accurately onEDTA decalcified bone. It isnoteworthy that the original reportby Rosenberg was carried out onfrozen sections of undecalcifiedarticular cartilage.2 In a recentpersonal communication (2003),Callis recommends the use ofnormal, undecalcified cartilagealong with an EDTA decalcifiedcartilage control when cartilagestudies are undertaken in order toappreciate the effects of EDTAdecalcification on staining.
Tinctoral differences in SOFGstaining may be the result ofseveral factors includingdifferences between species,between articular cartilage andgrowth plate cartilage, anddifferences in stain protocols,as well as the effects of particularfixatives and decalcifying agents.Therefore, if consistent tinctoralquality is required, it is imperative
Fig. 4. SOFG on 5-micron section of 14% EDTAdecalcified rat knee growth plate cartilage. Distinctiveproteoglycan staining is present as denoted by the redareas. Note the tinctoral differences within the samespecies between the two EDTA protocols. 20X
Fig. 5. SOFG on 5-micron section of 5% formic aciddecalcified rat knee growth plate cartilage. Distinctiveproteoglycan staining is present as denoted by the redareas. Note the tinctoral differences between rat andmouse in similar zones in all figures. 20X
Fig. 6. SOFG on 5-micron section of 5% formic aciddecalcified rabbit growth plate cartilage. Note thetinctoral differences as compared to the previousfigures. 20X
S a y g o o d n i g h t t o o v e r n i g h t p r o c e s s i n g .
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to use the appropriate controlthat will take all these factorsinto account.
Posting questions on Histonet iseasy and often tempting for thosewho don’t want to weed through alot of reference material but whosimply need a quick, expedientanswer. However, an accurateunderstanding of the technical andchemical details may well belacking in the Histonet response.This may lead us to apply specificconcepts to other problems orsituations in an erroneous manner.The reader has a responsibility toquestion and understand theconcepts thoroughly in order toavoid incorrect conclusions or thedissemination of misinformation.
AcknowledgmentsI would like to acknowledge the following individualswho contributed technical information that led to theconclusions drawn in this article:Gayle Callis, Veterinary Molecular Biology,
Montana State University, Bozeman, MTVicki Kalscheur, University of Wisconsin
School of Veterinary Medicine, Madison, WILiu Lichu, MD, Research Associate,
Arkansas Children’s Hospital Research Institute,Little Rock, AR
Martin Ronis, PhD, Professor in Pharmacology and Toxicology, UAMS, Associate Director,Arkansas Children’s Nutrition Center,Little Rock, AR
Ralph Sanderson, PhD, Professor of Pathology,Arkansas Cancer Research Center,University of Arkansas for Medical Sciences,Little Rock, AR
Larry Suva, PhD, Director, Center for Orthopaedic Research, University of Arkansas for Medical Sciences, Little Rock, AR
Frances L. Swain, Center for Orthopaedic Research,University of Arkansas for Medical Sciences,Little Rock, AR
The author thanks Bill Hogue, Center for OrthopaedicResearch, UAMS, Little Rock, AR, for photographicand computer-related assistance.
References1. Kalscheur VL. Bone and cartilage changes in
osteoarthritis: a proteoglycan stain. Histologic.2001;34:14-15.
2. Callis G, Sterchi D. Decalcification of bone: literaturereview and practical study of various decalcifyingagents, methods and their effects on bone histology.J Histotech. 1998;21:49-58.
AbstractImmunohistochemical (IHC)staining techniques are describedthat utilize a polyclonal anti-Treponema pallidum spirochetesantibody with colloidal gold andhorseradish peroxidase detectionsystems to demonstrate spirochetesin formalin-fixed, paraffin-embedded tissue sections for lightmicroscopy. The immunostainingreaction for the colloidal golddetection system is visualized withimmunogold silver staining (IGSS)enhancement. The immunostainingreaction with the horseradishperoxidase detection system isvisualized using DAB (3, 3’-diaminobenzidine) and AEC(3-amino-9-ethylcarbazole).
The IHC techniques used todemonstrate the organisms exhibitmore specific staining thantraditional silver techniquesbecause only the organisms arebeing stained in the tissue, readilydistinguishing them from thebackground. Additional work isneeded to determine the usefulnessof these techniques in thehistopathology laboratory.
IntroductionTreponema pallidum is thecausative agent for syphilis. Thediagnosis of syphilis in tissuesamples is dependent upon theaccurate demonstration of the
T pallidum organism. Traditionally,demonstration of these organisms intissue sections for light microscopyis performed using silverimpregnation methods such asWarthin Starry or Steiner & Steiner.These methods are sometimes verycumbersome and do not alwaysgive consistent and reproducibleresults. We describeimmunohistochemistry (IHC)staining techniques that utilizea polyclonal anti-Treponemapallidum spirochetes antibody withcolloidal gold and horseradishperoxidase detection systems todemonstrate spirochetes informalin-fixed, paraffin-embeddedtissue sections for light microscopy.We found that with these methods,the organisms are readily identifiedwithout the nonspecific stainingthat is often present when stainingwith traditional silver stainingmethods.
Materials and MethodsFormalin-fixed, paraffin-embeddedspecimens that were known tocontain spirochete organisms weresectioned at 4-6 microns, placed onSuperfrost Plus™ slides (ErieScientific, Portsmouth, NH), anddried at 58�-60�C. Sections weredeparaffinized and hydrated todistilled water. A routinehematoxylin and eosin (H&E)stain and a modification of thetraditional Steiner & Steinersilver stain were performed. Heat-induced epitope retrieval (HIER)using citrate buffer pH 6.0 in avegetable steamer was performedon the sections stained with IHC.Sections were immunostained withrabbit anti-Treponema pallidumspirochetes (Biocare Medical,Walnut Creek, CA). Specificationsfor the antibody are detailed inTable 1. Colloidal gold detectionwas achieved using the HistogoldKit (Zymed Laboratories Inc.,San Francisco, CA), as well as ahorseradish peroxidase (HRP)conjugated universal polymer(MACH2) detection system(Biocare Medical, Walnut Creek,CA). Each was used according tothe manufacturer’s specifications.
14
Immunostaining reactions for thecolloidal gold detection systemwere visualized using immunogoldsilver staining (IGSS)enhancement. Immunostainingreactions for horseradishperoxidase detection systemswere visualized usingaminoethylcarbazole (AEC)and/or diaminobenzidine (DAB)chromogen substrates.Specifications for detection systemsand counterstains used are detailedin Table 2. The immunostains wereevaluated to determine whichprocedure allowed for easyrecognition of the organisms.
ControlsTo verify the specificity of lightmicroscopy staining with Steiner& Steiner and immunostainingreactions, tissue sections of ratsmall intestine known to containspirochete organisms (NewcomerSupply, Middleton, WI) wereincluded in each staining run.
Immunogold Silver Staining (IGSS)Technique to DemonstrateTreponema pallidum Spirochetes
Formalin-fixed, paraffin-embeddedtissue sections are cut at 3-5 µ,placed on Superfrost Plus™ slides,dried at 58º-60ºC, deparaffinized,and hydrated to distilled water.Use chemically cleaned glasswareafter step 6.
1. Perform heat-induced epitoperetrieval (HIER) by placingsections into citrate buffer pH 6.0,and heating to 90ºC for 45 minutesin a vegetable steamer. Allowsections to return to roomtemperature before continuing.
2. Rinse in several changes ofdistilled water and then place inphosphate buffered saline (PBS).
3. Apply normal goat serum (NGS)to suppress nonspecificbackground staining.
15
Fig. 1. Steiner & Steiner silver stain; spirochete organisms are stained black but the background is often noisy,making organism identification difficult. 400X
Fig. 2. Immunogold IHC stain with silver enhancement and nuclear fast red counterstain. Organisms are stainedblack against a pink background. 400X
8. Apply silver enhancementsolution according tomanufacturer’s instructions.
Incubate: 5 minutes
NOTE: Monitor silverdevelopment microscopically. If notdark enough, rinse slides in PBSand apply fresh silver enhancementsolution for another 5 minutes.
9. Rinse well with distilled water.
10. Counterstain as desired:metanil yellow or nuclear fastred (NFR).
11. Dehydrate, clear, and mount inPermountTM (Fisher Scientific).
Stain Results: Spirochetes appearcrisp and black against a yellowbackground if counterstained withmetanil yellow, or against a redbackground if counterstained withNFR.
Formalin-fixed, paraffin-embeddedtissue sections are cut at 3-5 µ,placed on Superfrost Plus™ slides,dried at 58º-60ºC, deparaffinized,and hydrated to distilled water.
16
Fig. 3. IHC staining with horseradish peroxidase detection and DAB chromogen. Hematoxylin counterstain. 400X
Fig. 4. IHC staining with horseradish peroxidase detection and AEC chromogen. Hematoxylin counterstain. 400X
1. Perform heat-induced epitoperetrieval (HIER) by placingsections into citrate bufferpH 6.0, and heating to 90ºC for45 minutes in a vegetablesteamer. Allow sections to returnto room temperature beforecontinuing.
2. Rinse in several changes ofdistilled water.
3. Place sections in 3% H2O2
solution for 10 min to blockendogenous peroxidase. (Preparein methanol or distilled water.)
4. Rinse in several changes ofdistilled or deionized H2O andthen place in PBS.
5. Apply normal goat serum (NGS)to suppress nonspecificbackground staining.
Incubate: 20 minutes
6. Remove excess NGS fromaround sections and apply rabbitanti-Treponema pallidumantibody.
Incubate: Overnight in arefrigerator at 4ºC
7. Wash sections in PBSthree times.
8. Apply HRP Universal Polymer.
Incubate: 45 minutes
9. Wash slides in PBS three times.
10. Apply chromogen substrate:DAB or AEC.
Incubate: until desired colorintensity
11. Wash in several changes ofdistilled H2O.
12. Counterstain in hematoxylin.
13. Rinse in several changes ofdistilled H2O; intensify nuclearstaining with a bluing agent.
14. Rinse in several changes ofdistilled H2O. (If AEC is used,mount with aqueous mountingmedium; if DAB is used,dehydrate, clear, and mount inPermount.™)
Stain Results: Spirochetes usingAEC chromogen stain red againsta blue background. Spirochetesusing DAB chromogen stain
brown-black against a bluebackground.
ResultsAs seen in Figs. 2, 3, and 4, IHCstaining exhibits more specificstaining of the organisms thanthe traditional silver impregnationtechniques shown in Fig. 1. Onlythe organisms are stained in theimmunostained preparations andthey are readily distinguishedfrom the background. Whencounterstained with metanil yellow,the sections utilizing the IGSStechnique closely resemble thosestained with traditional silverimpregnation techniques.
DiscussionFor this investigation, a modifiedSteiner & Steiner silverimpregnation technique, and twoIHC techniques (HRP polymer andIGSS techniques) were performedon known positive T pallidumcontrols, as well as on spirochetecontrol slides that were purchasedfrom Newcomer Supply.Information from the vendorindicates that their spirochetecontrol slides are produced undercarefully controlled conditions inwhich T hyodysenteriae
17
TABLE 1ANTIBODY SPECIFICATIONS
Antibody Source Lot # Animal Dilution SpecialTreatment
Treponemapallidum Biocare 060201 Rabbit Prediluted HIER
TABLE 2DETECTION SYSTEMS/CHROMOGEN
Detection System Source Lot # Animals Chromogen Counterstain
Polymer (HRP) Biocare 031203 Universal M, R AEC, DAB Hematoxylin
Metanil YellowColloidal Gold Zymed 20973430R Rabbit N/A Nuclear Fast
Red (NFR) vl, k
Sometimes once is not enough.
That’s why Sakura features theHistoLogic® Archives on itsweb site at www.sakuraus.com.Whether you want to reviewrecent advances or decades-oldinnovations in histology, youcan find ample material inour archives.
The HistoLogic® Archivesenable users to access articlesfrom past HistoLogic® issuesdating back to 1971. Just typein a keyword in our archivesearch engine or look up anarticle by subject category.It’s that simple.
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microorganisms purchased fromAmerican Type Culture Collectionwere introduced into harvestedrodent organs.
Incubation time, temperature, andHIER to unmask the antigen wereoptimized for IHC staining.Consequently, sections underwentepitope retrieval using citratebuffer pH 6.0 and then wereincubated with the primaryantibody overnight at 4�C. Theantibody used in the IHCtechniques was a polyclonal rabbitanti-Treponema pallidum antibody.
The HRP polymer technique wasused to localize the organismsfollowed by AEC and DABchromogen substrates, before beingcounterstained with a modifiedMayer’s hematoxylin. Thespirochetes stained red with theAEC substrate and brown withthe DAB substrate. In tissuesections that were immunostainedusing the IGSS technique tolocalize and visualize thespirochetes, the organisms stainedblack against a pink-redbackground when using NFRas a counterstain, and against ayellow background when usingmetanil yellow as a counterstain.
ConclusionThe IHC staining techniques usedin this investigation seem to besuperior to traditional silverstaining methods for demonstratingT pallidum spirochetes. The IHCtechniques are reproducible andconsistent, and they exhibit morespecific staining than traditionalsilver staining techniques.Sensitivity is increased since thereis excellent contrast betweenstained organisms and background,allowing organisms to readily standout from the surrounding tissue.Additional work is needed in thehistopathology laboratory todetermine the usefulness of thesetechniques in the diagnosis ofspirochete disease.
AcknowledgmentsWe are most appreciative for the assistance of thefollowing students in the UTMDACC Program inHistotechnology: Patricia Amman, Gabriel Ayala,Shashi Reddy, and Olga Rodriguez.
References1. Garvey W. Silver stains. J Histotechnol.
1996;19(3):203-209.2. Holgate CS, Jackson P, Cowen PN, Bird CC.
Immunogold-silver staining: new method ofimmunostaining with enhanced sensitivity.J Histochem Cytochem. 1983;31:938-944.
3. Lucocq JM, Roth J. Colloidal gold and colloidal silvermetallic markers for light microscopic histochemistry.In: Techniques in Immunocytochemistry. Vol. 3.Academic Press: New York, NY; 1985:203-236.
4. Shi SR, Gu J, Kalra KL, Chen T, Cote RJ, Taylor CR.Antigen retrieval technique: a novel approach toimmunohistochemistry staining on routinelyprocessed tissue sections. Cell Vis. 1995;2:6-22.
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