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Histopathology for Animal Experiments Center for Laboratory Animal Research Samsung Biomedical Research Institute Apr 27, 2015 Jehoon Yang
Histopathology for Animal Experiments
Center for Laboratory Animal Research
Samsung Biomedical Research Institute
Apr 27, 2015
Jehoon Yang
Break the ice: smart (intellectual) animals
Data cooking (i)
Data cooking (ii)
Contents
1. Tissue sampling & fixation
2. Histological processing & Staining
3. Introduction of Immunohistochemistry
4. Non-invasive in vivo histopathology
César Baldaccini (1921-1998, French)
Histological tissue samples in SMC (i)
Histological tissue samples in SMC (ii)
Histological tissue samples in SMC (iii)
Tg/+ Tg/+ +/+ +/+
Tg/Tg Tg/+ Tg/+ +/+
Tg/+ +/+
P N 1 2 3 4 5 6 7 8 9 10
PSEN1
APP
HMONWMONMON
Axial Coronal Sagittal
In vivo experiment
Tissue Cassette
Histopathology (compound of three Greek words: ἱστόςhistos "tissue", πάθος pathos "diseases-suffering", and -λογία -logia) refers to the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides. In contrast, cytopathology examines free cells or tissue fragments.
from Wikipedia
What’s the Histopathology?
HISTOLOGY = Microscopic Anatomy
Paraffin Section
☞ Tissue sampling
☞ Fixation
☞ washing
☞ dehydration
☞ paraffin embedding
☞ slide sectioning
☞ staining
☞ Microscopic examination
Frozen Section
☞ Tissue sampling
☞ Frozen fixation
☞ slide sectioning
☞ staining
☞ Microsopy
Histopathological processing
Frozen vs. Paraffin section, rabbit vx2 tumor, H&E
Paraffin section, x100Frozen section, x100
The sooner the better
The smaller the better
The more the better
登高自卑
üTissue harvest and fixation are the first and most critical steps in the histological preparation process on which the quality and reproducibility of all of the subsequent steps are dependent.
Tissue sampling in SMC (i)
Tissue sampling in SMC (ii)
Tissue sampling in SMC (iii)
Purpose of fixation
The purpose of fixation is to preserve a sample of biological material (tissue or cells) as close to its natural state as possible in the process of preparing tissue for examination.
First, a fixative usually acts to disable intrinsic biomolecules – particularly proteolytic enzymes – which would otherwise digest or damage the sample.
Second, a fixative will typically protect a sample from extrinsic damage.
Finally, fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability.
Fixatives are used to preserve tissue from degradation, and to maintain the structure of the cells inclusive of sub-cellular components such as cell organelles (e.g., nucleus, endoplasmic reticulum, mitochondria).
Fixation should be performed immediately since delay permits autolysis and drying
Fixative ingredients
LIQUIDS
1. Absolute alcohol
2. Cold acetone
3. Formalin
4. Glutaraldehyde
5. Trichloroacetic acid
6. Acetic acid
1. SOLIDS
2. Mercuric chloride
3. Potassium dichromate
4. Osmium tetroxide
5. Picric acid
To summarize, although certain fixatives have special uses, phosphate
buffered neutral formalin is the most versatile and practical fixative and is
recommended for routine as well as many specialized procedure.
Laboratory Methods in Histotechnology Armed Forces Institute of Pathology
Formaldehyde
• Formaldehyde fixes tissue by cross-linking the proteins, primarily
the residues of the basic amino acid lysine.
• Other benefits include: long term storage and good tissue penetration.
• It is particularly good for immunohistochemistry techniques.
10% NBF (neutral buffered formalin)
• It is usually used as a 10% Neutral Buffered Formalin (NBF), that is aprox. 3.7% formaldehyde in phosphate buffered saline. Because formaldehyde is a gas at room temperature, formalin-formaldehyde gas dissolved in water (~37% w/v)-is used when making the former fixative.
± 4% formaldehyde = 10% formalin
± 8% formaldehyde = 20% formalin
• Paraformaldehyde is a polymerised form of formaldehyde, usually obtained as a fine white powder, which depolymerises back to formalin when heated.
Autolysis vs. NBF filtration rate
• Medawar: K = 5.5 for formaldehyde.
• Using this value NBF would penetrate 27.5 mm in 25 hours.
• Baker chose a gelatin/albumen gel to more closely mimic solid
tissue and determined that K = 3.6 for formaldehyde, or 18 mm in
25 hours.
§ 1 hour = 3.6 mm
§ 4 hours = 7.2 mm (1.8mm/hr)
§ 9 hours = 10.8 mm (1.2mm/hr)
§ 16 hours = 14.4 mm (0.9mm/hr)
§ 25 hours = 18 mm (0.72mm/hr)
§ 100 hours = 36 mm (0.36mm/hr)
Factors Affecting Fixation
pH
Should be kept in the physiological range, between pH 4-9. The pH for the ultrastructure preservation should be buffered between 7.2 to 7.4
Osmolarity
Hypertonic solutions give rise to cell shrinkage.
Hypotonic solutions result in cell swelling and poor fixation.
Size of the Specimen
1-4 mm thickness
Volume of the Fixative
At least 15-20 times greater than tissue volume
Temperature
Increasing the temperature will increase speed of fixation. However care is needed not to cook the specimen.
Duration
As a general rule 1hr per 1mm
ü Formalin fixed tissues for immunohistochemistry should be limited to 24-48 hours to maintain antigenicity.
Target and Chemical Fixative Do's and Don'ts
Target Fixative of Choice Fixative to Avoid
Proteins Neutral Buffered Formalin, Parafomaldehyde
Osmium Tetroxide
Enzymes Frozen Sections Chemical Fixatives
Lipids Frozen Sections*, Glutaraldehyde/Osmium
Tetroxide
Alcoholic fixatives, Neutral Buffered Formalin
Nucleic Acids Alcoholic fixatives, HOPE Aldehyde fixatives
Mucopolysaccharides Frozen Sections Chemical fixatives
Biogenic Amines Bouin's~, Neutral Buffered Formalin
Glycogen Alcoholic based fixatives Osmium Tetroxide
Frozen Sections preserve RNA and Lipids despite poor morphology. Compare to Paraffin sections, synonymous to Chemical Fixatives in the table, which destroy RNA and affect some antigens BUT give good morphology.
Pre fixation
PERFUSION FIXATION (i)
• fixation via blood flow
• For many purposes adequate fixation is obtained by simple immersion of small tissue pieces into the fixative solution.
• However, a more rapid and uniform fixation is usually obtained if the fixative solution is perfused via the vascular system, either through the heart or through the abdominal aorta.
• The fixative is injected into the heart with the injection volume matching cardiac output.
• The fixative spreads through the entire body, and the tissue doesn't die until it is fixed.
• This has the advantage of preserving perfect morphology
Perfusion fixation (ii)
Materials
1. Anaesthetic2. Scissors, forceps, and clamps for
surgical procedures3. Small forceps with fine claws4. Scalpel5. Vials (5-10 ml) with lids for specimens6. 0.9% saline7. 500 ml beakers8. 4% paraformaldehyde, fixation solution9. Gloves, eye goggles10. Perfusion pump (or flask with fixative
placed upside down about 150 cm above the operating table)
11. Short syringe needle for heart perfusion of aorta, length about 50 mm, outer diameter 1.3 - 1.5 mm
12. Perfusion set with drip chamber as used for intravenous blood infusions
Perfusion fixation: w/o, w perfusion
1. Sample thickness: 2 ~ 4 mm
2. Vol. of fixative: 15 ~ 20 times of tissue vol.
1. Dehydration
2. Clearing
3. Infiltration
Histopathological processing (i)
Histological processing
• Once the tissue has been fixed, it must be processed into a form in which it can be made into thin microscopic sections.
• Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated with paraffin.
• First, the water from the tissues must be removed by dehydration. This is usually done with a series of alcohols, say 70% to 95% to 100%.
• The next step is called "clearing" and consists of removal of the dehydrant with a substance that will be miscible with the embedding medium (paraffin).
• The commonest clearing agent is xylene. Toluene works well, and is more tolerant of small amounts of water left in the tissues, but is 3 times more expensive than xylene. Chloroform used to be used, but is a health hazard, and is slow.
• Finally, the tissue is infiltrated with the embedding agent, almost always paraffin.
Histopathological processing (ii)
1 2 43 5
Tissue atrophy after tissue processing
Tissue Processing
Special staining
Immunohistochemistry
Immunology
(antigen-antibody reaction)
Chemistry
(molecular reaction)
Histology
(tissue and organ)
• Immunohistochemistry (IHC) is an important application of monoclonal as well as polyclonal antibodies to determine the tissue distribution of an antigen of interest in health and disease.
• IHC refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
• Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a color-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine.
• IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.
Immunohistochemistry
The structure of rabbit IgG
• as an indication of tumor type:
- HMB45 protein expression in melanomas
- TTF-1 expression in primary pulmonary adenocarcinoma
• as an indication of metastasis:
- PSA (prostate specific antigen) in bone tumor
• as an indication of tumor aggression:
- Ki-67/M30
• as an aid in the assessment of patients considered for specified treatment
- HER2/neu: herceptin therapy
- c-Kit: Glivec in GIST
- EGFR: Erbitux in coloretal cancer
• as an indication of infectious process
• as a ……………………………………………………………………
Applications of Immunohistochemistry
CD31
10/6/14 BP5-4, x100
45
140508-7 (skin), x200
4-3: macrophage
H&E, x12.547
Lt2-1- lower iris, lateral plane
Insulin, x200
CD31, x200
double IHC: doxorubicin + HIF -1 alpha
DXR, x20048
HIF-1 alpha, x400
ü Deparaffin
ü Antigen retrival
ü Peroxidase blocking
ü Protein blocking
Three-Step LSAB Method (Labeled Streptavidin-Biotin)
ü Primary Ab
ü Secondary Ab-biotinylated
ü Streptavidin-HRP
ü Chromogen
ü Counter stain
Two-Step Polymer Method (Envision detection system, DAKO)
ü Deparaffin
ü Antigen retrival
ü Peroxidase blocking
ü Protein blocking
ü Primary Ab
ü Secondary Ab-HRP conjugated dextran polymer
ü Chromogen
ü Counter stain
Antigen Retrieval Techniques (i): HIER
Heat-induced Epitope Retrieval (HIER)• HIER is believed to reverse some cross-links and allows for restoration of
secondary or tertiary structure of the epitope. The protocol must be optimized for each tissue, fixation method, and antigen to be studied.
• In general, HIER has a much higher success rate than PIER.
• HIER is performed using microwave ovens, pressure cookers, vegetable steamers, autoclaves, or water baths.
• The heating length of 20 minutes appears to be the most satisfactory and the cooling usually takes about 20 minutes.
• Citrate buffer of pH 6.0 is the most popularly used retrieval solution and is suitable for most of antibody applications. The TRIS-EDTA of pH 9.0 and EDTA of pH 8.0 are second most used retrieval solutions.
• For all HIER methods, slides must be cooled before commencing IHC/ICC incubations. HIER is especially time-, temperature-, buffer-, and pH-sensitive, and the best method must be determined empirically.
Protease-induced Epitope Retrieval (PIER)• In the PIER method, enzymes including Proteinase K, Trypsin, and Pepsin have been
used successfully to restore the binding of an antibody to its epitope.
• Proteinase K is effective enzyme digestion reagent for membrane antigens such as Integrins, CD31, vWF, etc
• The mechanism of action is thought to be the cleavage of peptides that may be masking the epitope.
• The disadvantages of PIER are the low success rate for restoring immunoreactivity and the potential for destroying both tissue morphology and the antigen of interest.
• Therefore the optimal enzyme concentration and incubation time need to be tested.
• Combination of Heat Mediated and Proteolytic Enzyme Method is an alternative approach to unmask antigens if other methods did not work. It is especially useful when performing double or triple labeling of two or more antigens simultaneously.
Antigen Retrieval Techniques (ii): PIER
MoM (mouse on mouse) blocking (i)1. Mouse on Mouse Polymer IHC Kit (ab127055)
2. F(ab’)2 fragment secondary antibody (IHC an application guide, page.18)
3. Blocking of endogenous IgG using unconjugated AffiniPure Fab fragment Anti-Mouse IgG (H+L) (abcam protocol, Mouse on mouse staining procedure)
• We haven't compare these three method in details. However, I think method 1 (using mouse on mouse polymer IHC kit - ab127055) is the most convenient way to reduce background in mouse on mouse staining experiment because customer can simply follow the steps stated in the protocol booklet to finish the experiment.
MoM blocking (ii)
w PCNA, x400
w/o primary ab
w/o secondary ab
PCNA
Immunohistochemical expression of amyloid beta in AD-tg mice brain tisseues (brain serial section, LASB method)
Primary ab Amyloid beta
manufacture Covance Abcam
description Mouse monoclonal Rabbit polyclonal
Ab dilution 1 : 200 1:100
incubation 60 min RT 60 min RT
double IHC: amyloid beta + GFAP
Non-invasive In Vivo Histopathology
What’s the MOLECULAR IMAGING?
百聞不如一見!!!
Seeing is believing!!!
"In God we trust;
all others bring data"
He helped to create the field of molecular biology, proposed the concept and coined the term "molecular disease;“ founded the science of ortho-
molecular medicine, and was an activist for peace.
Linus Carl Pauling (1901-1994),
What is the Molecular Imaging?
• Molecular imaging is a new biomedical research discipline enabling the visualization, characterization, and quantificationof biologic processes taking place at the cellular and molecular levelswithin intact living subjectsincluding patients.
The society for molecular imaging
• To develop technologies and assays for imaging targeted macromolecules, and molecular and cellular events in living organisms
• To lead to better methods for studying biological processes as well as diagnostics and managing diseases
• The ultimate outcome of this technique should be for early diagnosis, and pre-disease state therapeutic response at the molecular level
The GOALS of the field are;
Translational Research: bench-to-bedside
Things to be considered:1. Spatial & temporal resolution2. Depth3. Sensitivity4. Type of molecular probe5. Perturbation of biological system
Detection range of imaging modalities
MRI/MRS/fMRI
Anatomic MR
Gross morphology
Specific target
Functional MR
Function
Activity
Molecular MR
Target imaging
Probe development
Metabolic MR
Tissue functionality
MRI/MRS/fMRI
• Noninvasive
• No limitation of depth
• Repetitive (no irradiation, stable)
• Near-cellular resolution (25~100 micron)
micro MR scanner
Nikola Tesla (Serbian Cyrillic: Никола Тесла; 10 July 1856 – 7 January 1943) was a a Serbian-American inventor, mechanical engineer, and electrical engineer.
Nucleus Spin QuantumNumber
Gyromagnetic
Ratio (MHz/1T)
Relative Sensitivityat Constant Field
Natural Abundance(%)
1H 1/2 42.58 1 99.8
13C 1/2 10.71 0.02 1.1
31P 1/2 17.25 0.06 100
23Na 3/2 11.26 0.09 100
19F 1/2 40.05 0.83 100
7Li 3/2 16.55 0.29 92.58
39K 3/2 1.99 0.0005 93.2
Gyromagnetic Ratio
Molecular imaging probe
Synthesis of HMON
Fig. 6. T2*-weighted MR imaging of the rat brain with MCAO treatment and enhanced by Fe3O4-PEG-PAEA10. The polymeric micelles were dissolved in the acidic area of the ischemic brain and the Fe3O4 nanoparticles were accumulated over time (arrows).
Molecular imaging probe
Chem. Commun., 2011, 47, 9176-9178
봄날은 간다. 허진호 감독
WHAT IS fMRI?
• Functional magnetic resonance imaging (fMRI) is a relatively new procedure that uses MR imaging to measure the tiny metabolic changes that take place in an active part of the brain (Ogawa, et al, 1990 a and b, 1992, 1993; Belliveau, et al, 1990, 1991).
• Functional MRI is based on the increase in blood flow to the local vasculature that accompanies neural activity in the brain.
• Since deoxyhemoglobin is paramagnetic, it alters the T2* weighted magnetic resonance image signal
6 months later
In vivo magnetic resonance spectroscopy
In vivo MRS
MRS provide a noninvasive window into brain
• In vivo magnetic resonance spectroscopy (MRS) directly measures chemically specific information.
• It is the only noninvasive technique for measuring concentration of metabolites from the living brain.
• Our MRS development at 11.7 Tesla, the highest field strength at which in vivo MRS has ever been attempted, has allowed, for the first time, detection of GABA turnover in vivo.
• In addition to the static levels of metabolites and metabolic fluxes measured by proton or 13C MRS, our recent discovery of 13C magnetization (saturation) transfer effect of specific enzyme reactions has made it possible to probe the action of several enzymes in vivo, pointing to new directions in 13C MRS technology development and applications.
In vivo evidence for reduced cortical glutamate-glutamine cycling in rats treated with the antidepressant/antipanic drug phenelzine
Yang J and Shen J. Neuroscience 135: 927 – 937 (2005)
Metabolitea Group A Group B
alanine** 0.05 ± 0.09 1.04 ± 0.38
aspartate 2.85 ± 0.27 2.61 ± 0.30
creatine 3.39 ± 0.15 3.52 ± 0.25
GABAc** 1.02 ± 0.17 2.30 ± 0.26
glutamated** 10.22 ± 0.30 8.53 ± 0.28
glutamined** 5.17 ± 0.31 4.58 ± 0.34
lactate 0.59 ± 0.18 0.50 ± 0.24
myo-inositol 4.52 ± 0.41 4.78 ± 0.33
N-acetylaspartate 10.50 ± 0.31 10.12 ± 0.40
phosphocreatine 5.11 ± 0.15 4.97 ± 0.25
phosphorylethanolamine
2.31 ± 0.56 2.12 ± 0.33
taurine 4.94 ± 0.31 4.73 ± 0.17
p Among 12 metabolites, Glu, Gln, GABA and Ala were the only metabolite which showed statistically significant changes.
p As a result, we became the first group in the world to detect turnover of the major inhibitory neurotransmitter GABA in the brain in vivo.
BW: 160 – 210 g
± Bruker microimaging spectrometer (Bruker Biospin, Billerica, MA) interfaced to an 11.7 Tesla 89-mm bore vertical magnet (Magnex Scientific, Abingdon, UK)
Animal preparation – 13C labeled glucose or acetate infusion
0
5
10
15
20
25
0 60 120 180 240 300
Time (min)
Blo
od
glc
(m
M/L
)
Control
ACZ
[2,5-13C]glucose; 2-13C FE: 0.99, 5-13C FE: 0.97, Cambridge Isotope Lab.
An initial bolus of 162 mg/kg/min of 1.1 M [1-13C]glucose in the first 10 minutes followed by constant-rate infusion of the same glucose solution at 62.8 mg/kg/min i.v.) was performed.
Detection of reduced GABA synthesis following inhibition of GABA transaminase using in vivo magnetic resonance signal of [13C]GABA C1.
Non-human primate experiment
Surface coil and cannulation for Monkey MRS
In vivo 13C magnetic resonance spectroscopy of human brain on a clinical 3 T scanner using [2-13C]glucose infusion and low-power stochastic decoupling.
100 Years of Research on Alzheimer’s Disease
Ongoing study
Molecular Pathogenesis of AD
Detection of reduced GABA synthesis following inhibition of GABA transaminase using in vivo magnetic resonance signal of [13C]GABA C1
Prospective results
Thank you.
