Unit 9 Common Problems Encountered in IHC

What happens if primary antibody is not compatible with the secondary antibody?

no staining

What happens if chromogenic substrate is not compatible with the enzyme used?

no staining

What happens if reagents are in the incorrect order?

no staining

What happens if little or no antigen in the tissue?

weak or no staining

What happens if destruction of antigenicity by excessive heat?

weak or no staining

What happens if used old, expired, inactive reagents?

weak or no staining

What happens if HIER or EIER insufficient or omitted?

weak or no staining

What happens if alcohol based counterstain is used on AEC, Fast red, Fast blue, or other alcohol soluble chromogens?

weak or no staining

What happens if primary antibody incubation time is insufficient?

weak or no staining

What is prozone effect?

primary antibody is too concentrated and prevents any antibody from binding

What happens if there is Prozone effect?

weak or no staining

What happens if excessive counterstaining masks staining reaction?

weak or no staining

What happens if excessive buffer is left on sections, resulting in diluted reagents?

weak or no staining

What happens if there is use of sodium azide in peroxidase reactions?

weak or no staining

What happens if there is the use of PBS in alkaline phosphatase reactions?

weak or no staining

What happens if antibody titer is too high?

overstaining

What happens if incubation is too long?

overstaining

What is cross-reactivity?

the specific binding of an antibody to an antigen (epitope) other than the antigen of interest

What is quenching?

inactivation of chemical activity by an excess of reactants or products

What is used to quench endogenous alkaline phosphatase?

levamisole

T/F endogenous peroxidase is only a problem with HRP detection systems

true

What is used to quench endogenous peroxidase?

3% hydrogen peroxide

What is used to prevent endogenous human immunoglobulins?

commercially prepared secondary antibodies that are preabsorbed to human serum proteins

What is a problem if ABC or LSAB detection systems are used?

endogenous biotin

What can unmask biotin?

HIER

__ can be used to bind endogenous biotin

Free avidin

Endogenous activity is reduced in __ fixed tissue

formalin

T/F free antigen in necrotic specimen does not show excessive background staining

false

What does diffusion of antigen lead to?

specific staining in the wrong place

What does phagocytosis of target antigens result in?

specific staining in the wrong place

What happens if tissue was allowed to dry with reagents on?

non-specific staining

T/F contaminating antibodies is more common in monoclonal

false

What happens if there is non-immunological binding of immune sera by hydrophobic and electrostatic forces to certain sites within tissue sections, such as connective tissue?

background staining

Remedy for: incubation of sections in blocking reagent, such as an immunoglobulin that will not react with the primary (usually species that secondary is raised in)

background staining

Remedy for: the charged protein molecules of the serum will bind to the charged areas on the tissue section, preventing antibody reagents from binding to these areas non-specifically

background staining

What can be used for blocking?

powdered milk or casein

What is addition of detergents to the wash buffers a remedy for?

background staining

__ act to reduce hydrophobic interactions

detergent

How to remedy endogenous pigment (melanin or hemosiderin)?

• use chromogen that will contrast the endogenous pigment

• bleach/stain melanin

What is used to remove mercury pigment?

iodine/sodium thiosulfate

How to prevent undissolved granules of chromogen or counterstain?

proper preparation and storage of reagents

What is used to get rid of formalin pigment?

alcoholic picric acid

What does levamisole block?

endogenous alkaline phosphatase (except GI alkaline phosphatase)

What is used to block endogenous alkaline phosphatase?

levamisole

After completion of IHC procedure, there is no staining of the positive control. Alkaline phosphatase enzyme was used with DAB chromogen. What caused the negative staining?

enzyme is not compatible with the chromogen

In an HRP detection system, what is the function of H2O2 in methanol?

blocks endogenous peroxidase

Polyclonal antibody serum often labels non-specific antigens in tissue. Identify the likely reason

polyclonal antiserum contains many antibodies

Skin stained four keratin. HRP was used with routine AEC. After coverslipping with synthetic mounting medium, AEC stain is not seen. What would you do to avoid this?

coverslip with aqueous mounting medium

Spleen stained using a monoclonal LCA and a LSAB system with DAB. There’s non-specific staining all over the section. Likely cause?

endogenous biotin

IHC stained tissues share non-specific reaction of granulocytes with chromogen and substrate. Likely cause?

endogenous peroxidase wasn’t blocked

In IHC, what is the function of casein?

prevents background staining of proteins

What can Bouin solution be used to block?

endogenous alkaline phosphatase

Most likely cause of antigen diffusion

necrosis