Unit 6: IHC Techniques

T/F for direct method, only one antibody is used

true

T/F for direct IHC method, the primary antibody is used to locate the antigen

true

T/F fluorescent IHC methods have superior sensitivity

false

T/F chromogenic IHC methods lack sensitivity

true

T/F most detection systems today use indirect IHC methods

true

T/F for indirect IHC method, the primary antibody is used to locate the antigen

false

T/F for indirect IHC method, the secondary antibody is used to locate the antigen

true

IHC indirect method locates epitopes on the __ of the primary antibody

FC

Which animal is often used for secondary antibody production (in indirect method)?

goat

T/F only one secondary antibody can combine with the primary antibody

false

Secondary antibodies can be labeled with _ or _ for visualization

fluorochrome, enzyme

For soluble enzyme immune complex techniques, what must be directed against Igs of the species of the primary and the enzyme immune complex?

secondary antibody

Both PAP and APAAP require the use of __ for visualization

chromogens

What chromogens are used for PAP?

DAB, AEC

What chromogens is used for APAAP?

new fuchsin, fast red, fast blue

Each avidin molecule can bind __ molecules of biotin

4

T/F LSAB is more sensitive than ABC

false

What is good for ultrastructure immunolocalizatoin?

immunogold method

For double stain, what must be used for primary antibody?

mouse monoclonal and a rabbit polyclonal/monoclonal cocktail

T/F PBS can be used with double stain

false

Is indirect or direct IHC more sensitive?

indirect

T/F in polymer procedures, the secondary antibody step is unlabeled

false

What is tertiary antibody directed against?

species that the secondary antibody was raised in

Primary antibody is a polyclonal rabbit antibody. What is the secondary antibody?

goat anti-rabbit

T/F HRP can be an antigen

true

In PAP unlabeled antibody method, the primary antibody was raised in rabbit. Identify the species of the PAP complex.

rabbit

Regarding the avidin-biotin complex IHC method, which is true?

The ABC complex binds to biotin-labeled secondary antibody

This method of IHC detection uses a labeled primary antibody

direct

When performing the APAAP IHC procedure, the anti-ALP was raised in mouse. What’s the species of the primary antibody?

mouse

T/F secondary antibodies are polyclonal

true

The primary antibodies, mouse CD3 (T cells) and rabbit L26 (B cells), are used in a cocktail for double staining. The secondary anti-mouse is labeled with HRP and secondary anti-rabbit is labeled with AP. DAB and FR are used as chromogens. What color do the T cells stain?

brown

Another term for soluble enzyme immune complex techniques

unlabeled antibody methods

For LSAB ALP, what is the primary antibody conjugated with: streptavidin, biotin, AP, or FR

none of the above

For LSAB, what is conjugated with avidin?

biotin

T/F in multiplex staining, 2 different epitopes can be demonstrated using the same chromogen

true

T/F ALP can function as an antigen

true

What is this?

polymer

Double staining was done using sequential reactions, with elution after the first chromogen. What enzyme is recommended for first reaction?

HRP

T/F avidin is vitamin H

false

What does the secondary antibody react with on the primary antibody?

both heavy and light chains of the primary antibody

Why is streptavidin better than just avidin?

It contains no carbohydrates and is uncharged at neutral pH, producing less background staining