IMMUNOHISTOCHEMISTRY PART 1- LN

IMMUNOHISTOCHEMISTRY

Makes use of antigen antibody reactions by directly labeling of the antibody or by means of a secondary labeling method

IMMUNOHISTOCHEMISTRY

Involves the identification of tissue or cellular antigens or phenotypic markers

Phenotypic markers

found in tissues

Antibodies

used as detectors

IgG

most common form of antibody used in IHC

POLYCLONAL

• Produced by different cells

• It can react with various epitopes

POLYCLONAL

May be obtained from laboratory animal sources

POLYCLONAL

• Main source: rabbits (immunized in order to produced Ab)

• Other sources: goats, pigs, sheep

MONOCLONAL

• More specific

• It produces only 1 type of Ab

• It can react only with 1 specific type of epitope

MONOCLONAL

It is produced from individual clone of plasma cells

MONOCLONAL

Animal source: mice

ENZYMES

• most widely used enzyme label is HORSE RADDISH PEROXIDASE

chromogen

If HORSE RADDISH PEROXIDASE is used, we need to use ________ (color developer)

brown

Diaminobenzidine (DAB)

brick red

3-amino-9-ethylcarbazole (AEC)

ALKALINE PHOSPHATASE

Alternative to HRP:

Hematoxylin

ENZYMES traditional counterstain

FLUOROCHROME LABEL

• Involves the use of FLUORESCEIN ISOTHIOCYANATE (FITC)

• use of Fluorescence Microscope

30-60 minutes at room temperature

ENZYMES Optimum incubation time to link the Ab with enzyme peroxidase:

LECTINS

Aside from the use of antibodies to detect antigens, lectins can also be used.

LECTINS

are plant or animal proteins which can bind to tissue carbohydrate

CRYOSTAT FROZEN SECTIONS

Fixed in a few seconds using absolute methanol or acetone

CRYOSTAT FROZEN SECTIONS

Purpose of fixation:

• To prevent destruction of labile antigenic sites

• To preserve the position of antigens

PROCESSED SPECIMENS

• Formalin fixed specimen spx

• paraffin embedded spx

Antigen retrieval

If the specimens for IHC are processed specimens, this will require _____________.

Proteolytic enzyme retrieval (PIER)

• most common method

• Commonly used enzymes are - trypsin or protease

Microwave antigen retrieval / Heat Induced Epitope retrieval

Duration is 20 minutes

Pressure cooking antigen retrieval

Not preferred because it is time consuming

POSITIVE CONTROL

A tissue section with the antigen being detected

NEGATIVE CONTROL

T o prepare the negative control, we omit primary antibody from the staining schedule

INTERNAL TISSUE CONTROL

a.k.a. BUILT IN CONTROL

INTERNAL TISSUE CONTROL

It contains the target antigen, not only in the tissue element, but also in adjacent normal tissue elements