IMMUNOHISTOCHEMISTRY: or IHC is the localization of antigens in tissue sections by the use of labeled antibody as specific reagents through antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold. It is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. It is also used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.
ANTIGEN: is a substance that stimulates antibody response. It has many antigenic sites on its surface called epitopes which have the capacity of binding to the antibody. ANTIBODY: is defined as an immunoglobulin (Ig) capable of specific combination with the antigen. It is produced in response to the invasion of an antigen in the body. It consists of two heavy chains and two light chains, and named according to its heavy chains; e.g. IgG has gamma type heavy chains and IgA has alpha light chains.
MONOCLONAL ANTIBODY: Binds to specific antigen. Monoclonal antibodies are generally considered to exhibit greater specificity than polyclonal antibodies. POLYCLONAL ANTIBODIES: are a heterogeneous mix of antibodies that recognize several epitopes of one antigen. They are made by injecting animals with peptide antigens, and then after a secondary immune response is stimulated, isolating antibodies, against different epitopes of the antigen, from whole serum.
TECHNIQUES OF IHC STAINING DIRECT
The direct method is a one-step staining method, and involves a labeled antibody reacting directly with the antigen in tissue sections. This technique utilizes only one antibody and the procedure is therefore simple and rapid. However, it can suffer problems with sensitivity due to little signal amplification and is in less common use than indirect methods.
DIRECT IHC STAINING METHOD
TECHNIQUES OF IHC STAINING INDIRECT
The indirect method involves an unlabeled primary antibody (first layer) which reacts with tissue antigen, and a labeled secondary antibody (second layer) which reacts with the primary antibody. This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. The second layer antibody can be labeled with a fluorescent dye or an enzyme.
INDIRECT IHC STAINING METHOD
TECHNICAL ASPECTS OF IHC STAINING1)
FIXATION AND SECTIONING:
To ensure the preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential. However, inappropriate or prolonged fixation may significantly diminish the antibody binding capability. In general, many antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections. The development of antigen retrieval techniques further enhanced the use of formalin as routine fixative for immunohistochemistry in many research laboratories.
TECHNICAL ASPECTS OF IHC STAINING
Some antigens will not survive even moderate amounts of aldehyde fixation. Under this condition, tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat. The sections should be kept frozen at -20C or lower until fixation with cold acetone or alcohol. After fixation, the sections can be processed using standard immunohistochemical staining protocols. The disadvantage of frozen sections includes poor morphology, poor resolution at higher magnifications, special storage needed, limited retrospective studies and cutting difficulty over paraffin sections.
TECHNICAL ASPECTS OF IHC STAINING2) TISSUE ADHESIVES:
In case of IHC, the tissue section is liable to fall off due to frequent rinsing of the slides, use proteolytic enzymes or microwave. This problem can be avoided by using positively charged slides or coating the slides with adhesives e.g. poly-L-lysin, albumin or histogrip.
TECHNICAL ASPECTS OF IHC STAINING3) ANTIGEN RETREIVAL: The demonstration of many antigens can be significantly improved by the pretreatment with the antigen retrieval reagent that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites. The techniques involved the application of heat for varying lengths of time to formalin-fixed, paraffin-embedded tissue sections in an aqueous solution (commonly referred to as the retrieval solution). This is called "Heat Induced Epitope Retrieval (HIER)". Another method uses enzyme digestion and is called "Proteolytic Induced Epitope Retrieval (PIER)".
TECHNICAL ASPECTS OF IHC STAINING4) BLOCKING:
Background staining may be specific or non-specific. Inadequate or delayed fixation may give rise to false positive results due to the passive uptake of serum protein and diffusion of the antigen. Such false positives are common in the center of large tissue blocks or throughout tissues in which fixation was delayed. Antibodies, specially polyclonal antibodies, are sometimes contaminated with other antibodies due to impure antigen used to immunize the host animal. The main cause of non-specific background staining is non-immunological binding of the specific immune sera by hydrophobic and electrostatic forces to certain sites within tissue sections. This form of background staining is usually uniform and can be reduced by blocking those sites with normal serum.
TECHNICAL ASPECTS OF IHC STAININGBlocking of these unwanted reactions is done by:a)
Peroxidase block is done by incubation of tissue sections for 10-20 minutes with 3% hydrogen peroxide in methanol. Protein block is done by incubation for 10-20 minutes with normal animal serum. Biotin block (only in tissues containing biotin as liver, kidney and brain) is done by incubation for 10-20 minutes with avidin solutions
TECHNICAL ASPECTS OF IHC STAINING5) STAINING: A) PAP Method (peroxidase anti-peroxidase method): PAP method is one development of the indirect technique and it involves a third layer which is a rabbit antibody to peroxidase, coupled with peroxidase to make a very stable peroxidase anti-peroxidase complex. The complex, composed of rabbit gaba-globulin and peroxidase, acts as a third layer antigen and becomes bound to the un-conjugated goat anti-rabbit gabaglobulin of the second layer. The sensitivity is about 100 to 1000 times higher since the peroxidase molecule is not chemically conjugated to the anti IgG but immunologically bound, and loses none of its enzyme activity. It also allows for much higher dilution of the primary antibody, thus eliminating many of the unwanted antibodies and reducing non-specific background staining.
TECHNICAL ASPECTS OF IHC STAININGB) Avidin-Biotin Complex (ABC) Method:
ABC method is standard IHC method and one of widely used technique for immunohistochemical staining. Avidin, a large glycoprotein, can be labeled with peroxidase or fluorescein and has a very high affinity for biotin. Biotin, a low molecular weight vitamin, can be conjugated to a variety of biological molecules such as antibodies. This technique involves three layers. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is a complex of avidin-biotin peroxidase. The peroxidase is then developed by the DAB or other substrate to produce different colorimetric end products.
TECHNICAL ASPECTS OF IHC STAININGC) Labeled Strept-Avidin Biotin (LSAB) Method: Strept-avidin, derived from streptococcus avidini, is a recent innovation for substitution of avidin. The strept-avidin molecule is uncharged relative to animal tissue, unlike avidin which has an isoelectric point of 10, and therefore electrostatic binding to tissue is eliminated. In addition, strept-avidin does not contain carbohydrate groups which might bind to tissue lectins, resulting in some background staining. LSAB is technically similar to standard ABC method. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is enzyme-strept-avidin conjugates to replace the complex of avidin-biotin peroxidase. The enzyme is then visualized by application of the substrate chromogen solutions to produce different colorimetric end products. The third layer can also be Fluorescent dyestrept-avidin such as FITC-Strept-avidin if fluorescence labeling is preferred.
TECHNICAL ASPECTS OF IHC STAININGMultiple Labeling: It is often useful to be able to stain for two or more antigens in one common tissue section. This can be achieved by:
Immuno-fluorescence method using different fluorescent dyes. Peroxidase conjugated antibodies developed with different chromogen substrates to produce the end products of different colors.
TECHNICAL ASPECTS OF IHC STAINING6) CHROMOGEN: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 catalyze a color-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as FITC, rhodamine, Texas Red or Alexa Fluor.
IHC STAINING PROCEDURE USING ABC1) Circle tissue section with a diamond pen and keep at 50C overnight. 2) Deparaffinization, (2 changes of xylene). 3) Rehydration, (descending concentrations of alcohol). 4) Antigen retrieval. 5) Peroxidase block. 6) Protein block. 7) Primary antibody application for one hour (minimum), then wash with PBS. 8) Secondary biotin-conjugated antibody for 30 minutes (minimum), then wash with PBS. 9) Strept ABC/HRP complex for 30 minutes (minimum), then wash with PBS. 10) Chromogen for 6-10 minutes (maximum), then wash twice with water. 11) Counterstaining in hematoxylin for 30-60seconds, then wash twice with water. 12) Dehydration in ascending concentrations of alcohol, then mount.
CONTROLS OF STAINING1)
Negative control: stained sections following all steps of IHC except for the step of antibody application which is replaced by non-immune serum or even PBS. It is done in every run to avoid interpretation of any non-specific staining (seen also in the negative control) as specific. Positive control: a stained section known to be positive for the antigen following all steps of IHC, exactly as the specimen. It is done in every run to verify the validity of the technique and the quality of the reagents.
H&E-stained section which is serial to the immunostained one should be examined to be aware of the morphology of the tissue section under test. True positive staining should be localized in its correct site i.e. membranous, cytoplasmic, nuclear or stromal. Homogenous pale brown or yellow staining of cells is non-specific. No staining should be demonstrated in the negative control. The positive control should show true positive immunostaining.
NUCLEAR IMMUNOSTININGER nuclear staining A- benign adenosis B- Ductal carcinoma
P63 nuclear immunostaining
Cytoplasmic immunostaining of metalloproteinase in prostatic carcinoma
MEMBRANOUS IMMUNOSTAINING Her-2
immunostaining in breast cancer