INTRODUCTION
IHC is a technique for identifying cellular or tissue constituents by means of antigen antibody interactions.
It involves localisation of the antigens in the tissue sections by the use of labeled antibody through ag-ab interactions that are visualized by a marker such as flourescent dye, enzyme,radioactive element or colloid gold.
In ambiguous areas of histopathological diagnosis, IHC provides a way of identifying substances in tissues. It has emerged as powerful tool which can provide supplemental information to the routine morphological assessment of the tissues.
HISTORY OF IHC
1941-ALLERT H COONS developed immunofluroscence technique ,described new way of visualizing tissue constituents using a ab labeled with flouroscent dye.The first dye to be used was Fluorescein isocyanate [ apple green fluoroscence] 1966-Nakane & Pierce developed enzyme labeling
PRINCIPLE
Specific antigens in the tissues are localized based on the ag and ab recognition . The site of ab is identified either by tagging the antibody directly or indirectly with a visible label.The visual marker may be a fluorescent dye,colloidal metal,hapten,radioactive marker or enzyme.
ANTIGEN-a molecule that induces the formation of an antibody. Bears one or more ab binding sites. These sites are highly specific topographic regions known as epitopes ANTIBODIES- they are immunoglobulins found in blood ,tissue fluids and many secretions. 5 types-IgA,IgD,IgE,IgG,IgM. IgG is the commonest and used in IHC .
-Each ab composed of 2 heavy and 2 light chains. -They are formed in the humoral immune system by the B-lymphocytes after recognition of a foreign antigen.Terminal region of each arm varies in a sequence and is known as variable domain. This variability provides specificity for a particular epitope and enables the ab to bind specifically to ag against which it was raised.
ANTIBODY-ANTIGEN BINDING The amino acid side chain of the variable domain of of an antibody form a cavity which is geometrically and chemically complementary to a single type of antigen epitope. The ag and ab are held together by a combination of hydrogen bonds , electrostatic and vander waals forces.
Antibody
types:
Poyclonal antibodies: Heterogeneous population of antibodies produced against several epitopes of a single antigen. More sensitive but less specific Monoclonal antibodies: Homogenous population of antibodies for a single epitope. More specific but less sensitive
Polyclonal antibodies are produced by immunizing an animal with a purified specific molecule bearing the antigen of interest . Humoral response to the immunogen numerous clones of plasma cellseach clone produces antibodies to variety of epitope present on the immunogen Antibodies harvested by bleeding the animal to obtain immunoglobulin rich serum
Monoclonal antibodies are produced as follows: .Mouse is injected with a purified antigen produces antibody against it. .When large amounts of antibodies are produced the mouse is sacrificed and spleen containing large amounts of B lymphocyte is removed .B cell suspension is mixed with myeloma cells in a medium that will cause the cell to fuse
.The resultant is a hybridoma.(hybridomafusion of myeloma cells and B cells of the same species) .Hybridoma is tested for the clone antibody against the desired epitope .Required hybridoma is grown in tissue culture, the supernatant fluid contains the antibody produced by the hybridoma
IHC METHODS
Direct technique
One step method Labeled antibody reacts with antigen in tissue sections (Label conjugated may be a flurochrome or an enzyme) Advantages: rapid, short and easy Disadvantages: poor signal amplification, hence less sensitive Need to manufacture labeled ab against every ag to be tested
Uses: Demonstration of Ig & complement in frozen sections of skin and renal biopsies.
Direct Method
Labeled Antibody
Tissue Antigen
Advantages
. More sensitive than the direct method . More versatile method, as a number of primary abs can be used with same conjugated secondary ab.
Secondary Antibody
Primary Antibody
Tissue Antigen
PEROXIDASE ANTIPEROXIDASE METHOD .Peroxidase antiperoxidase complex(rabbit)-3 peroxidase molecule and 2 antiperoxidase antibodies. .They are bound to the unconjugated primary antibody(rabbit anti human antibody) by a second layer of bridging antibody(swine antirabbit ab).
.Secondary antibody is applied in excess so that one of its 2 identical binding sites binds to the primary ab and other to rabbit PAP complex
Alkaline phosphatase anti alkaline phoshphatase method(APAAP) .Principle is same as PAP method except that PAP complex is replaced by APAAP complex.
Advantages:
Increase in sensitivity,100 to 1000 fold increased signal amplification .
Avidin-biotin conjugate method(ABC) .Relies on the strong affinity of avidin or streptavidin to vitamin biotin (nonimmunological) .3 step technique1st layer- unconjugated primary antibody 2ndlayer- biotinylated secondary antibody (against species of primary animal) 3rd layer- complex of peroxidase conjugated biotin and avidin
. Biotin cojugate with biological molecule such as antibodies and enzymes. . Avidin has 4 binding sites for biotin. . Free sites of avidin molecule allows binding to biotin molecule on the secondary antibody.
ABC Method
(avidin-biotin complex method )
Disadvantages .Avidin (glycoprotein) has tendency to bind to lectins in the tissue- non specific staining. .Avidin has isoelectric point =10, at neutral pH it is +vely charged hence binds non specifically to vely charged structures. .Tissues rich in endogenous biotin (liver, kidney)will require blocking of endogenous biotin before applying primary antibody by preincubation with avidin.
.Streptavidin is isolated from streptomyces avidinii. .Has higher affinity for biotin. .Has isoelectric pH=7,and not a glycoprotein hence reduced non specific staining.
SP Method
(streptavidin peroxidase conjugated method)
Advantages
Increased sensitivity Allows higher dilution of the primary antibody
Conjugation of both anti-mouse and anti rabbit secondary antibodies enables the same reagent to be used for both monoclonal and polyclonal primary antibodies Advantages .Increased sensitivity .Minimized non-specific background staining .Reduction in the total number of assay steps
Other methods
Immunogold silver staining technique Hapten labeling technique Mirror image compliment antibody labeling technique(MICA) Biotinylated tyramide signal amplification
Neutral buffered formalin is the fixative most commonly used. Cross linkage of proteins formed by the formalin can mask the antigenic site if over fixed. Fixation time, pH, temperature can affect IHC reactions. Formalin should be fresh and buffered to pH of 7 to 7.6
Formalin
Zinc Formalin
shorter fixation times; minimal need for antigen unmasking or retrieval; preserves better tissue Ag morphology shorter fixation times; better cryostat sections (cold EtOH/acetone); good preservation of cytoplasmic intermediate filaments
Alcohol/Acetone
variable; often occurs in tissue processing secondary to formalin fixation; 10-15 minutes for cryostat sections/cytology smears 1-12 hrs.
Bouin
fixes tissues rapidly; useful particularly for endocrine tissues and tumors
decreased preservation of many types of Ags, particularly lipidcontaining Ags; longer fixation may result in brittle tissues; poor penetration may result in under-fixation; special handling and disposal requirements tissue hardening; surface Igs not well demonstrated; special handling and disposal requirements
B-5
1-6 hrs.
primary use in lymph node tissue; enhanced cytologic detail and immunoreactivity of cytologic immunoglobulin (Ig), intracytoplasmic Ags.
Paraffin embedding temperature should be maintained between 56 and 60.c Sections should be mounted on tissueadhesive coated slides sections which are not flat with non adherent ridges are likely to be digested or torn off during antigen retrieval or washing. .Poly-l-lysine .3-aminopropyltriethoxysilane .Electrostatically coated slides
Staining
Antigen retrieval:
. Most antigens are partially or completely masked after formalin fixation due to cross linking of the amino acid group. Prior to immunostaining, sections are pretreated to unmask the antigens. . Methods: -Enzyme epitope retrieval -Heat induced antigen retrieval (HIER) -Combination of HIER & enzyme retrieval
Enzyme epitope retrieval: .Trypsin / chymotrypsin method .Protease method .Pepsin method .Pronase or proteinase method -Period of 5 to 15 min @ 37.c -Enzyme treatment is believed to cleave the protein cross link .
Heat induced antigen retrieval: .Relies on application of heat to tissue sections in an aqueous medium (retrieval solution). .Commonly used retrieval solutions. Citrate buffer at pH 6 EDTA at pH 8 TRIS-EDTA at pH-9 .Degree of immunoreactivity restored depends on the duration of incubation(10 min to 1hr) and attained temperature.
Various methods of heating .Microwave processing .Pressure cooker .Water bath / steamer .Autoclave
Combination of HIER and enzyme retrieval method : .Alternate approach if other methods do not work .Useful when double or triple labeling for 2 or 3 antigens
MCA-497 staining of Liver tissue fixed in NBF for 28 days, with citrate buffer & heat-mediated antigen retrieval
Endogenous enzymes blocked prior to staining -to eliminate false positive reactions RBCs WBCs and muscle contain abundant peroxidase activity. Blocked by preincubation of section with absolute methanol containing 3% H2O2 . Other methods0.3% H2O2 .with 0.1%Na azide in PBS /TBS Glucose oxidase Acid hematin
BLOCKING ENDOGENOUS ALKALINE PHOSPHATASE ACTIVITY In paraffin sections lost in processing In frozen sections blocked with 1mM concentration levamisole
Proteins in the serum occupy charged sites on tissue sections. This serum neither interferes nor participates in immunological reactions during staining procedure.
(a) Pan-cytokeratin, mouse thymus. Left: Perithymic mediastinal tissues exhibit profound nonspecific background staining (brown). Right: GSH abolishes nonspecific background retaining thymic epithelial cell labeling . (b) c-myc, ffpe mouse liver. Left: Staining diffusely present in hepatocytes, including in tumor nodule (arrow). Right: GSH coincubation reveals that c-myc from the albumin-promoted transgene is concentrated in
(c) Pan-cytokeratin, ethanol-fixed mouse colon. Left: Nonspecific fluorescent signal (green) in colon wall and mesenteric adipocytes (arrows). Right: GSH coincubation enhances specific fluorescent visualization of colonic epithelium
Antibody standardization
For optimal staining it is necessary to use antibodies at correct dilutions Not the darkest staining but greatest contrast between the specific +ve staining and unwanted background non specific staining
Diluent for antibody - Freshly prepared Tris-buffered saline(TBS) or autoclaved TBS at 4.c -Bovine serum albumin(BSA) 0.1% .Titrations performed using manufacturers antibody titers by using different antigen retrieval methods and buffers .Once ideal method of ag retrieval is identified antibody titer optimized using different dilutions. .Dilution of ab has to be worked with each new batch
.If antibody is concentrated, smaller aliquots made and stored at -20.c after standardizing appropriate dilution .Once aliquot vial is thawed it should not be frozen again, as freeze and thaw cycle results in loss of potency of ab .Use autoclaved tips for making dilutions.
Washing buffers
To prevent formation of ag- ab complexes that will precipitate onto sections -interpretation problems and background staining. It is necessary to remove the unbound ab before incubating the next layer Achieved by washing the sections with TBS between the antibody incubations TBS with surfactant tween 20 is most often used
Chromogens
To visualize the enzymes labelling the antibodies Enzyme substrate reactions - convert colorless chromogens into visible colored end-products 3,3-diaminobenzidine hydrocloride(DAB) Brown end product Insoluble in alcohol or other organic solvents,hence can be subjected to routine dehydration and permanent mounting
3 amino-9ethyl carbazole(AEC) .on oxidation produces rose red end product ,soluble in alcohol .Non alcoholic counter stain like mayers haematoxylin is used. .Aqueous mouting done with glycerol. .AEC is susceptible to oxidation,exposure to light hence dark storage
Other chromogens used .4-chloro 11 naphthol .Hanker-yates reagent .Naphthol AS MX phosphate .Fast red TR .Fast blue BB .New fuschin .Nitro-blue tetrazolium
Control
Positive control: is known to contain ag under question and should be handled in the same fashion as the test tissue Negative control: treated in same fashion as that for the test except for the primary ab which is omitted and replaced with non immune serum or wash buffer. .Used to assess specificity of the method and presence of background staining
Internal control
The ag being tested is present in the adjacent normal tissue. eg: normal breast in case of ca breast for ER/ PR.
Double staining Two or more different antigens can be demonstrated on cells. Involves incubation of section with one set of reagents to stain one ag followed by incubation with second set
erad
Double staining for kappa & lambda of reactive lymph node using sequential horseradish technique & alk phosphatase method
SUMMARY OF STEPS
Deparaffinize /rehydration Heat slides in oven at 65.c for 1 hr Deparaffinize/hydrate:2 xylene washes followed by 100%ethanol rinses ,followed by 95%ethanol, 70%ethanol, 50%ethanol, 30%ethanol, followed by water and TBS for 5 min Antigen retrieval Staining Wash slides with TBST for 5 min on a shaker Inactivate endogenous peroxidase by covering tissue with 3% H2O2 for 10 min
Wash slides 3 times with TBS Block slide with blocking solution for 1 hr Dilute primary antibody Apply primary antibody to each section and incubate overnight in humidified chamber(4.c) Wash slides 3 times with TBST Apply to each sections the diluted secondary antibody for 1 hr at room temperature Wash slides 3 times with TBST
Add freshly prepared DAB substrate to the sections Incubate sections with substrate at room temperature until suitable staining develops(2 -5 min) Rinse sections with water Counter stain with hematoxylin Rinse section with water Dehydrate samples using two rinses with 100%ethanol followed by 2 rinses with xylene Mount coverslip on slide
Fresh Tissue
Fixative
Paraffin ribbon
Deparaffinize
PBS
Wash
Chromogen development
Fix with the pre-cooled fixative for 5 -10 min, at room temperature Rinse 3-4 X in PBS. Continue with the immunohistochemical staining protocol. The absence of formalin eliminates the need for an antigen retrieval step. However, if frozen tissue or cytological specimens have been fixed in formalin, antigen retrieval can be attempted although the friable nature of the specimens may compromise the success.
Solution
Longer deparaffinization/ fresh xylene
Inactive antibodies or inactive ABC Replace with a new batch, check reagents for proper storage Ab concentration too low Inadequate ab incubation time Increase the concentration / check for optimal dilution of abs Increase the incubation time
Over staining
High concentration of Proper dilution primary or secondary ab Incubation time too long High incubation temperature Section dried out Reduce the time
High background
Inadequate washing of sections Wash 2 -3 times between steps
Endogenous enzymes Blocking using 3%H2O2 peroxidase, alk in methanol/ levamisole phosphatase in tissue prior to primary ab incubation Endogenous biotin Block agent prior to activity in tissue primary ab incubation
Non specific binding of primary antibody Non specific binding of secondary antibody Diffusion of tissue ags due to inadequate fixation Sections dried out
Use high dilutions of primary antibody Treat with normal serum from same species as secondary antibodies Increase duration of postfixation Use humid chamber
T H A N K Y O U