Immunhistochemistry

 
Background

Immunhistochemistry is a powerful technique that allows a researcher to detect molecules within a tissue microenvironment. Protein expression can be studied in relation to tissue or cell compartment, and changes due to either normal or pathologic stimuli can be evaluated. Either direct or indirect methods can be used to detect an antibody bound to an antigen. The direct method uses a labeled reagent, such as a fluorescent tag or an enzyme-linked secondary antibody that binds to the primary antibody. A biotinylated secondary antibody can be used for better sensitivity allowing a tertiary step such as an enzyme-linked avidin or streptavidin to react. While the indirect method involves more assay steps, the amplification process at each step leads to increased sensitivity.



General procedure
· Mount formalin fixed paraffin embedded sections (4 micron) onto silane-coated slides.
· Air dry slides at 37ºC for 24 h.
· Deparaffinize and rehydrate.
· Incubate specimens for 20 minutes in 1-3 drops of serum block. Aspirate serum from slides.
· Immediately add 1-3 drops of pre-diluted primary antibody.
· Incubate for 2 hours. Rinse with PBS then wash in PBS twice for 2 minutes each on a stir plate. Aspirate excess liquid from slides.
· Incubate for 30 minutes in 1-3 drops of biotinylated secondary antibody. Wash as above.
· Incubate for 30 minutes in 1-3 drops of HRP-streptavidin complex. Wash as above.
· Add 1-3 drops HRP substrate mixture. Develop for 30 seconds-10 minutes, or until desired stain intensity develops. Rinse with deionized H2O and transfer to a deionized H2O wash for 2 minutes on a stir plate.
· Counterstain, dehydrate and mount slides
Negative controls underwent similar staining procedure, but with the exclusion of the primary antibody. Positive controls were carcinomas that were shown to be immunoreactive for the studied antigens in preliminary studies.


Interpretation of staining results


The presence of immunostaining in carcinoma cells was evaluated by two independent researchers without knowledge of the clinical outcome. The presence and extent of staining was evaluated, using the following scale: 0, no staining; 1, staining of 1-5 % of tumor cells; 2, staining of 6-25 % of tumor cells; 3, staining of 26-50 % of tumor cells and 4, staining of > 50 % of tumor cells. Staining of both cell membrane and cytoplasm was interpreted as positive.