Immunohistochemistry

Antigen

‭ A‬‭ molecule‬‭ which‬‭ induces‬‭ the‬‭ formation‬‭ of‬‭ an‬ antibody‬‭ and‬‭ bears‬‭ one‬‭ or‬‭ more‬‭ antibody‬‭ binding‬ sites.‬

‭ These‬‭ are‬‭ highly‬‭ specific‬‭ topographical‬‭ regions‬ composed‬‭ of‬‭ a‬‭ small‬‭ number‬‭ of‬‭ amino‬‭ acids‬‭ or‬‭ monosaccharide‬‭ units‬‭ known‬‭ as‬‭ antigenic‬ determinant groups of epitopes.

Antibody

belong‬‭ to‬‭ the‬‭ class‬‭ of‬‭ serum‬‭ proteins‬‭ known‬‭ as‬‭ immunoglobulins.‬‭

They are found in blood and tissue fluids, as well as many sections such as tears and saliva.

Antibody-antigen binding

are‬‭ held‬ together‬‭ by‬‭ a‬‭ combination‬‭ of‬‭ hydrogen‬‭ bonds,‬ electrostatic interactions, and van der Waals’ forces.

Antibody specificity

This‬‭ is‬‭ the‬‭ characteristics‬‭ of‬‭ an‬‭ antibody‬‭ to‬‭ bind‬ selectively to a single epitope on an antigen.

Avidity

Is‬‭ a‬‭ related‬‭ property‬‭ referring‬‭ to‬‭ the‬‭ heterogeneity‬‭ of‬ the‬‭ antiserum‬‭ which‬‭ will‬‭ contain‬‭ various‬‭ antibodies‬ reacting‬‭ with‬‭ different‬‭ epitopes‬‭ of‬‭ the‬‭ antigen‭ molecule

Affinity

Is‬‭ the‬‭ three-dimensional‬‭ fit‬‭ of‬‭ the‬‭ antibody‬‭ to‬‭ its‬ specific‬‭ antigen,‬‭ and‬‭ is‬‭ a‬‭ measure‬‭ of‬‭ the‬‭ binding‬ strength‬‭ between‬‭ the‬‭ antigenic‬‭ epitope‬‭ and‬‭ its‬ specific antibody-combining site.

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Sensitivity

This‬‭ is‬‭ the‬‭ relative‬‭ amount‬‭ of‬‭ antigen‬‭ which‬‭ an‬‭ IHC‬ technique‬‭ is‬‭ able‬‭ to‬‭ detect.‬‭

Gig

Antibody that is most commonly used in IHC

Epitope

The structural part of the antigen that reacts with the antibody

Histochemistry

Science‬‭ that‬‭ combines‬‭ the‬‭ technique‬‭ of‬‭ biochemistry‬ and‬‭ histology‬‭ in‬‭ the‬‭ study‬‭ of‬‭ chemical‬‭ constitution‬‭ of‬ tissues and cells.

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Immunology

Is a science that deals with the immune system, Cell Mediated and humoral aspect of immunity and immune responses.

Immunohistochemistry

Is the localization of a known antigen in tissues by utilizing antibodies towards a specific antigen.

Fixation

Prevents elation, degradation, and modification of antigens.

Preserves position of antigens.

Provides target for antibody molecules.

Formaldehyde

Is the preserve fixative

Slide Preparation

• 2-4 micron are cut onto slide

• The tissues are further adhered to the slide by heating.

• Deparaffinization

Deparaffinization

Tissue is treated in a series of xylene and alcohol to remove paraffin.

Antigen Retrieval

• Enables partial reversal of formaldehyde induced conformational change of antigen

• Increases the accessibility of Ab to the zag

• Enzyme Digestion

• Heat

2 Methods of Antigen Retrieval

Ag removal

Choice of _________depends on the Ag to be demonstrated

Heat Induced Epitope Retrieval (HIER)

Widely used in Antigen removal

• Proteolytic Enzyme Digestion

• Heat-induced Epitope Retrieval

• Blocking

Antigen Retrieval Techniques

Pre-Treatment: Proteolytic Enzyme Digestion

Purpose : breaks formalin cross-links to expose masked antigenic sites.

Heavy chain immunoglobulins, complement, cytokeratin detection.

Proteolytic Enzyme Digestion is used for?

Trypsin and Protease

Common Enzymes under Proteolytic Enzyme Digestion

• Deparaffinize and rehydrate sections‬

• Block‬‭ endogenous‬‭ peroxidase‬‭ (0.5%‬‭ methanol‬‭ in H2O2, 10-15mins)

• Rinse in water and distilled water

Pre-Treatment steps (Proteolytic Enzyme Digestion is)

‬‭- 0.1% trypsin + 0.1% calcium chloride, pH 7.8‬

‬‭ - Preheat slides and solution at 37℃‬

‬- Digest, then stop in cold water

Proteolytic Enzyme Digestion Trypsin Method

• 0.05 -0.1% protease in distilled water, pH 7.8

• Shorter digestion time due to stronger activity

Proteolytic Enzyme Digestion Protease Method

Pre-Treatment : Heat-Induced Epitope Retrieval

Purpose: reverses formalin-induced antigen masking in FFPE tissues

• Heat breaks protein cross-links to unmask epitopes

• Buffers (EDTA, citrate) chelate calcium, aiding cross-link reversal

Mechanism of Heat-Induced Epitope Retrieval

EDTA, Citrate

Common Buffers of Heat-Induced Epitope Retrieval

• Microwave

• Pressure cooker

• Steam and Water bath

Heating sources of Heat-Induced Epitope Retrieval

To prevent false-positive staining and ensure specific binding of antibodies to the target antigen

Goal of Blocking

• Peroxide Block

• Protein Block

2 main ways of Blocking

Peroxide Block

• Blocks endogenous peroxidases activity present in tissues especially in blood-rich organs like liver, spleen, kidney

3% H2O2

Inactivâtes the enzyme

Protein Block

• Blocks all non specific sites in tissue sections that antibodies Kay react to.

• Reduces background noise and improves clarity of specific antigen detection

3% Bovine serum albumin

10% Normal serum is used or protein like _______

• Polyclonal Antibodies

• Monoclonal Antibodies

Types of Primary Antibodies

Polyclonal Antibodies

Are produced by injecting an antigen into an animal, commonly a rabbit or a goal.

Monoclonal Antibodies

Are made using hybrids a technology where a single type of immune cell is fused with a cancer cell to create a cell line that produces only one specific antibody.

Polyclonal Antibodies

Are more sensitive but can have higher background noise because they bind to multiple sites.

Hybridomas

Monoclonal Antibodies are produced by?

Monoclonal Antibodies

Monoclonals‬‭ are‬‭ highly‬‭ specific‬‭ and‬‭ ideal‬‭ for‬‭ targeting‬ a single‬‭ epitope‬‭ which‬‭ helps‬‭ reduce‬‭ background‬‭ and‬ improves consistency.

Direct Technique

Primary antibody is directly conjugated to a detectable label (fluorochrome or horseradish peroxidase)

• Simple and rapid (single-step)

• Fewer reagents and incubation steps

• Less risk of background staining for secondary antibodies

Advantages of Direct Technique

• Low sensitivity‬

• Risk‬‭ of‬‭ false‬‭ negatives‬‭ for‬‭ low-antigen‬ targets‬

• Less‬‭ suitable‬‭ for‬‭ detecting‬‭ small‬‭ antigen‬ quantities‬

• Largely‬‭ replaced‬‭ by‬‭ more‬‭ sensitive‬‭ indirect‬ Methods.

Limitations of Direct Technique

Direct Technique

The earliest method developed in immunohistochemistry

• Primary Antibody

• Secondary Antibody

• Signal Amplication

Indirect Techniques

Primary Antibody

Unconjugated

Secondary Antibody

Labeled antibody that binds to the primary antibody

• Inexpensive

• High sensitivity via signal amplication

Advantages of Indirect Technique

Signal Amplication

Multiple secondary antibodies bind to different epitopes on primary antibody

Horseradish peroxidase (HRP)

Common Enzyme used in Indirect Technique

Ag-Ab conjugates

Are visualized by the use of labels.

Enzyme Labels

Are enzymes that produce a colored precipitate in the presence of a substrate.

Detection System

• Direct or single step method

• Indirect or two step method

Peroxidase

Most widely used label and produces a dark brown precipitate when Diamino Benzindine (DAB) is added

Alkaline Phosphatase

Is also used and produced either red or blue precipitates

Substate

chemical‬‭ compound‬‭ that‬‭ is‬‭ acted‬‭ upon‬‭ by‬‭ an‬‭ enzyme‬ used‬‭ in‬‭ IHC‬‭ (horseradish‬‭ peroxidase‬‭ or‬‭ alkaline‬ phosphatase)

Chromogène

colorless‬‭ compound‬‭ that,‬‭ when‬‭ oxidized‬‭ or‬‭ reacted‬‭ by‬ the‬‭ enzyme‬‭ and‬‭ its‬‭ substrate,‬‭ produces‬‭ a‬‭ colored‬ end-product visible under the microscope

Brown color

End product of Horseradish‬‭ peroxidase‬‭ + substrate‬ hydrogen‬‭ peroxide‬‭ or‬‭ chromogen‬ diaminobenzidine‬‭

Red color

End product Chromogen‬‭ amino‬‭ ethyl‬‭ carbazole

Blue color

End product of 4-chloro-1-naphthalene

Dark blue

Hanker-Yates reagent end product

Red-purple

Alpha-naphtol pyromania end product

Hematoxylin and Eosin

Most commonly used cpunterstain (gold standard in IHC)

Hematoxylin

Provides contrast to the primary stain.

• stains nucleic acid blue

Eosin

Stains eosinophilic strictures in shades of red, pink, and orange

Positive control

Cells or tissues that are known to contain the specific Ag.

• Detects false negative due to improper fixation and tissue processing.

Negative control

Omission of Primary Ab with the same tissue and procedure.

Useful to detect background staining caused by nonspecific binding

Deparaffinize sections longer or change fresh xylene

Inadequate deparaffinization .

solution?

Replace with a new batch of antibodies

Inactivate primary antibodies.

Solution?

Aliquot antibodies into smaller volumes and store in freezer (-20 to -70 C) and avoid repeated freeze and thaw cycles.

Antibodies do not work due to improper storage.

Solution?

Increase the concentration of antibodies. Or run a serial dilution test to determine the optimal dilution that gives the best signal to noose ratio.

Antibody concentration was too low

Solution?

Increase antibody incubation time

Inadequate antibody incubation time.

Solution?

Increase duration of post fixation or try different fixatives

Inadequate or improper tissue fixation.

Solution?

Reduce the duration of post- fixation or perform an appropriate antigen retrieval procedure

Tissue over-fixation

Use secondary antibody that will interact with primary antibody

Incompatible secondary and primary antibodies

Replace with a new batch of reagents

Inactive secondary antibody or other reagents

Increase substrate incubation time

Inadequate substrate incubation time

Choose a correct mounting medium

Incorrect mounting medium

Check notes or procedure used

Reagents applied in wrong order or steps omitted

Reduce antibody concentration or perform a titration to determine the optimal dilution for primary and secondary antibodies

The concentration of antibodies was too high

Reduce incubation time

Incubation time was too long

Reduce incubation temperature

Incubation temperature was too high

Reduce substrate incubation time

Substrate incubation time was too long

Avoid sections being dried out

Sections dried out

• Epithelial

• Intermediate Filament

• Neuroendocrine

• Germ cell

• Mesenchymal

Tumor Markers

• Keratin

• EMA (Epithelial Membrane Antigen)

• CEA ( Carcinoembryonic Antigen)

• TTF-1 ( Thyroid Transcription Factor - 1)

• PSA ( Prostate Specific Antigen)

Epithelial tumor markers

Keratin

Sensitive marker for epithelial tumors

Commonly found in carcinomas but can also stain some non-epithelial tumors like mesotheliomas or germ cell tumors.

CK7

Positive in lung, breast, uterus, ovary (serious tumors)

• Typically negative for CK 20

CK20

Positive in colon and stomach carcinomas

• negative for CK7

Bladder (Transitional Cell Carcinoma), mucinous ovarian tumors

Positive CK7 and CK20

Renal CC, Hepatocellular CC, Prostate, Thyroid, Squamous CC

Negative CK7 and CK20

Epithelial Membrane Antigen

• Positive in breast, lung, kidney adenocarcinomas

• Negative/weak in HCC, adrenal, embryonal carcinomas

• Negative in sarcomas, lymphomas, melanomas, meningiomas

Carcinoembryonic Antigen

Positive in GI, pancreas, lung, breast, Ovary, uterus, cervix

• Adeocarcinoma positive

• Mesothelioma negative

Negative in prostate, thyroid, kidney carcinomas

Commonly used in GI and other epithelial tumors

Thyroid Transcription Factor -1

• Positive in thyroid, lung adenocarcinomas, neuroendocrine tumors.

• Nuclear transcription factor that helps identify lung and thyroid carcinomas, including small cell lung cancer and neuroendocrine tumors.

Prostate Specific Antigen

• Diagnostic marker for prostatic adenocarcinoma

• Also seen in pancreatic and salivary gland tumors

• Specific marker for prostatic tissue, crucial in diagnosing prostate cancer.

• Can occasionally show up in pancreatic tumors

• Actin

• Vimentin

• Desmin

• Glial Fibrillary Acidic Protein

• Neurofilament

• S-100 Protein

Intermediate Filament Markers

Actin

Contractile protein in muscle and some non-muscle cells.

Marker for muscle tumors