Prior to the detection of the primary antibody in the immunohistochemistry technique, some other important experiment including but not limited to deparaffinization, antigen retrieval and blocking non-specific ionic binding must be carried out prior to primary antibody detection. We have previously looked at deparaffinization and rehydration of paraffin-embedded tissue, as well as antigen-retrieval protocol, permeabilization and blocking non-specific binding on the sectioned tissue sample on the slide. We also looked at antibody selection and staining protocol. See other sections on immunohistochemistry in this website for update. In this section, we look briefly on the primary antibody detection in immunohistochemistry staining technique. Prior to the antibody staining cum detection, it is also important to run a parallel control experiment using a control tissue sample. Performing a control staining experiment will help to ensure that the observed staining pattern during immunohistochemistry is authentic and specific, and not due to some unspecific binding or cross-reaction that may arise during the experiment. Both positive control and negative control should be run alongside the main experiment.
At this stage of antibody detection, either the monoclonal antibody or polyclonal antibody is used for the antibody detection analysis, depending on which type of antibody is required for the particular immunohistochemistry analysis.
The protocol for antibody detection is explained briefly below:
- After draining off the blocking buffer from the slide (as explained in previous section talking about blocking non-specific binding), add the appropriate volume (e.g. 100 µl) of diluted monoclonal or polyclonal antibody (known as the primary antibody) to the tissue section on the slide.
- Incubate the slide for about 45 min or 1 hour at room temperature. Ensure to keep slides carrying sectioned tissues in a humid environment, preferably in a humidified chamber or box. This is important because it will prevent the sections from drying out, a phenomenon that can lead to a false positive result or bad result.
- After incubation, wash tissue sections on the slides with PBS for 10 min at room temperature. This process should be done thrice.
- Add appropriate volume (e.g. 100 µl) of the biotinylated secondary antibody to the tissue sections on the slides.
- Incubate tissue sections on slide for about 30 min at room temperature. Slides should always be kept in a humidified chamber to prevent drying out.
- After incubation, wash slides again in PBS for 30 min. This step should be done twice.
- Apply a given volume of DAB reagent (e.g. 100 µl) to the tissue sections on the slide. This stage helps to reveal the colour of the antibody staining on the tissue sections. Watch the slides for about 5 min until the desired colour intensity is developed. DAB is carcinogenic; thus it should be handled with gloved-hands and in a hood. DAB = Diaminobenzidine tetrachloride.
- After the colour develops, wash the slides in PBS for 2-5 min. This step should be done twice.
- Counter stain the slides using nuclear fast red reagent or hematoxylin reagent for about 1-2 min. This step is usually done if nuclear counterstaining is required in the experimental plan.
- Rinse the slides carrying the tissue sections in running distilled water. THE NEXT STEP IS DEHYDRATION AND MOUNTING OF TISSUE SECTIONS ON SLIDES FOR VISUALIZATION TO SEE ANTIBODY-ANTIGEN COMPLEX FORMATION. SEE NEXT SECTION ON THIS WEBSITE FOR FURTHER EXPLANATION ON DEHYDRATION AND MOUNTING.
Positive control is the control tissue sample that is known to express the protein or antigen of interest. It will help the researcher to know that the immunohistochemistry experiment is working properly and as expected.
Negative control is the control tissue sample that is not known to express the protein or antigen of interest. This sample will help the researcher know that the observed staining pattern in the immunohistochemistry experiment is due to some specific signals.