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. 

Deparaffinization

Rehydration

Antigen retrieval

BlockingPrimary antibody incubatio

nSecondary antibody incubatio

nDetection Counterst

aining

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 stainingBefore 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