The basics of immunohistochemistry

Principle Anigen (protein of interest) Primary antibody Secondary antibody

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 IHC takes its name from the roots ▫" immuno," in reference to antibodies used in the procedure, ▫and "histo," meaning tissue (compare to immunocytochemistry). Immunohistochemistry is the localization of antigens or 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, or colloidal gold

TYPES OF DETECTION Visualising an antibody-antigen interaction can be accomplished in a number of ways. Enzymatic staining ▫an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction immunofluorescence ▫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. utilizes only one antibody procedure is short and quick. However, it is insensitive due to little signal amplification and rarely used since the introduction of indirect method. 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

IMP!! Primary antibody ▫Should be raised against the antigen of interest ▫E.g.  for detecting human antigens- raise antibodies in specie other than humans! ▫HOW?  Injecting human MYOSIN protein in an animal specie e.g rabbit  Antibodies will be raised against human antigen in rabbit  These will be called as RABBIT ANTI-HUMAN MYOSIN ANTIBODY

Secondary antibody ▫Detects the Fc portion of the primary ▫must be against the antibody of the animal species in which the primary antibody has been raised) ▫E.g.  In our example primary antibody was rasied in – rabbit  Secondary antibody should detect this rabbit made antibody  So it must be an ANTI-RABBIT  Must be raised in another specie (other than humans & rabbits)

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 microtome ▫Mounting ▫onto glass slides that are coated with an adhesive ▫ 3-aminopropyltriethoxysilane (APTS) or poly-L-lysine ▫gelatin, egg albumin or Elmer's glue.

DeparaffinizationRehydrationAntigen retrievalBlocking Primary antibody incubation Secondary antibody incubation DetectionCounterstaining 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

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 used ▫normal serum, non-fat dry milk, BSA or gelatin

Non-specific staining Before blockAfter 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