Immunohistochemistry (IHC) is the most common application of immunostaining. IHC exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells and tissue, most detected and examined with the light microscope.
--Blocking solution (e.g. 5% BSA, 1% milk, or 1% serum from the same species as the secondary antibody)
--Washing buffer (e.g. PBS or TBS)
--Detection system (e.g. DAB or fluorescent dye)
--Mounting medium (e.g. DPX or glycerol)
1) Deparaffinization and rehydration: Dewax the tissue sections by incubating them in xylene or a xylene substitute for 10 minutes, followed by rehydration through graded alcohol solutions (100%, 95%, 70%, and 50%) for 5 minutes each. Rinse the sections in running tap water for 5 minutes.
2) Antigen retrieval: Retrieve the antigenicity of the tissue by heating the sections in a pressure cooker with antigen retrieval buffer (e.g. 10 mM citrate buffer, pH 6.0) for 10-15 minutes, or in a microwave for 5-10 minutes. Let the sections cool down at room temperature for 20 minutes.
3) Blocking: Block non-specific binding of the antibodies by incubating the sections in blocking solution for 30 minutes at room temperature or overnight at 4°C.
4) Primary antibody incubation: Dilute the primary antibody in blocking solution according to the manufacturer's instructions, and incubate the sections with the primary antibody for 1 hour at room temperature or overnight at 4°C.
5) Washing: Wash the sections with washing buffer (3 times, 5 minutes each) to remove unbound primary antibody.
6) Secondary antibody incubation: Dilute the secondary antibody in blocking solution according to the manufacturer's instructions and incubate the sections with the secondary antibody for 30 minutes at room temperature.
7) Washing: Wash the sections with washing buffer (3 times, 5 minutes each) to remove unbound secondary antibody.
8) Detection: Detect the bound antibodies by using a detection system (e.g. DAB or fluorescent dye) according to the manufacturer's instructions.
9) Counterstaining: If desired, counterstain the sections with a nuclear counterstain (e.g. hematoxylin) for 5-10 minutes.
10) Dehydration and mounting: Dehydrate the sections through graded alcohol solutions (50%, 70%, 95%, and 100%) for 5 minutes each, followed by xylene or a xylene substitute for 10 minutes. Mount the sections with a mounting medium and coverslip.
11) Note: The above protocol is a general guideline and may need to be optimized for specific antibodies and tissues.