IHC in frozen tissues or in transfected cells
- Primary Antibody: incubate the tissue sections or cytospins preparations with 100 μl of primary antibody at the appropriate dilution for 30 min at room temperature.
- Wash the slides in 300 ml PBS for 5 min.
- Repeat the wash in fresh PBS.
- Secondary Antibody: incubate the sections with 100 μl of secondary antibodya (dilution 1:100) for 30 min at room temperature.
- Rinse the slides in 300 ml of PBS twice as above.
- Detection of antigen/antibody complexes: apply 100 μl DAB substrate solutionb to the sections to reveal the color of the antibody staining. It is important that the solution is freshly made just before use. Allow the color to develop for approximately 5-8 minutes until the desired color intensity is reached.
- Rinse the slides in 300 ml PBS twice as above.
- Counterstain the slides by immersing them in Hematoxylinc for 1-2 min.
- Rinse the slides in running tap water for 3 min.
- Repeat washing procedure twice.
- Mount the slides using a coverslip and a mounting agentd. The mounted slides can be stored at room temperature permanently.
a Polyclonal goat anti-mouse HRP secondary antibody (Dako P0447)
b Liquid DAB+Substrate Chromogen System (Dako K3468)
c Carazzi’s Hematoxylin solution DC (Panreac 255298.1610)
d Aquatex aqueous mounting agent (Merck 1.08562.0050)