The basics of immunohistochemistry

THE BASICS OF IMMUNOHISTOCHEMI

STRY

PRINCIPLE Antigen-antibody interactions

Antigen

Antibodies produced Antibodies binds antigen

HOW IT IS EMPLOYED? Antigen = Protein of our interest Antibody= we generate

TECHNIQUES BASED ON THIS PRINCIPLE ELISA

FACS

WESTERN BLOTTING

IMMUNOHISTOCHEMISTY

IMMUNOHISTOCHEMISTRY – WHAT’S GOOD ABOUT IT? Antibodies bind to antigen in specific

manner Can be used to locate particular cells

and proteins Can be used to identify cellular events –

e.g.apoptosis

INTRODUCTION Immunohistochemistry is the localization of antigens

(proteins) in tissue sections by the use of labeled antibodies as specific

reagents through antigen-antibody interactions visualized by a marker such as fluorescent dye,

enzyme.

IHC takes its name from the roots "immuno," in reference to antibodies used in the

procedure, and "histo," meaning tissue (compare

toimmunocytochemistry).

TYPES OF VISUALISATION Visualising an antibody-antigen interaction can be

accomplished in a number of ways. Chromogenic

an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction 

fluorescent Alternatively, the antibody can also be tagged to

a fluorophore, such as fluorescein or rhodamine 

WHAT CELLULAR ANTIGENS CAN WE TARGET?

Cytoplasmic Nuclear Cell membrane

APPLICATIONS disease diagnosis drug development and biological research

TYPES OF IHC Direct Indirect

DIRECT METHOD-PRIMARY ANTIBODY ONLY

one step staining method involves a labeled antibody reacting directly with the antigen in

tissue sections. This technique utilizes only one antibody and the procedure is

short and quick. However, it is insensitive due to little signal amplification and

rarely used since the introduction of indirect method.

Goat anti-actin labeled with 594

INDIRECT METHOD – PRIMARY AND SECONDARY ANTIBODIES involves an unlabeled primary antibody (first layer) which react

with tissue antigen, and a labeled secondary antibody (second layer) react with

primary antibody  This method is more sensitive due to signal amplification  economic

Goat anti-actin

Donkey anti-goat labeled with 488

IHC PROTOCOL

SAMPLE PREPARATION (FFPE) FORMALIN FIXED PARAFFIN EMBEDDED

Tissue fixation To ensure the preservation of tissue architecture and cell morphology,

prompt and adequate fixation is essential the most common fixative is formaldehyde (FF)Embedding in paraffin to maintain the natural shape and architecture of the sample during long-term storage and sectioning for

IHC (PE)Sectioning Into slices as thin as 4-5 μm with a microtomeMounting onto glass slides that are coated with an adhesive  3-aminopropyltriethoxysilane (APTS) or poly-L-lysine gelatin, egg albumin or Elmer's glue. 

Deparaffinization

Rehydration

Antigen retrieval

BlockingPrimary antibody incubatio

nSecondary

antibody incubatio

n

Detection Counterstaining

Mounting &

observation

ANTIGEN RETRIEVAL What?

Retrieve your antigen for detection by IHC

Why? Formaldehyde fixation generates methylene bridges that crosslink proteins in tissue samples; these bridges can mask antigen presentation and prevent

antibody binding.

How? to unmask the antibody epitopes, 1. either by heat (heat-induced epitope retrieval; HIER) 2. or enzymatic degradation (proteolytic-induced epitope

retrieval; PIER).

BLOCKING ENDOGENOUS TARGET ACTIVTIY What?

Quenching or masking endogenous forms of enzymatic proteins (biotin, peroxidases or phosphatases)

Why? When using Enzymatic detection To prevent false positive and high background

detection. How?

Hydrogen peroxide – peroxidases levamisole - Alkaline phosphatase Avidin - biotin

BLOCKING NON-SPECIFIC SITES What?

Masking sites that are similar to target sites Why?

antibodies may partially or weakly bind to sites on nonspecific proteins that are similar to target

nonspecific binding causes high background staining that can mask the detection of the target antigen. 

How?Commonly blocking buffers are usednormal serum, non-fat dry milk, BSA or

gelatin

NON-SPECIFIC STAINING

Before block After block

CONTROLS Postive control

to test a protocol or procedure and make sure it works. It will be ideal to use the tissue that has the expression of your

antigen If the positive control tissue showed negative staining, the

protocol or procedure needs to be checked until a good positive staining is obtained.

Negative control To test for the specificity of an antibody involved Exclude the primary antibody – no color should be obtained