EuroMAbNet: European Monoclonal Antibodies Network

Protocols

IHC in frozen tissues or in transfected cells

Procedure

• 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 antibody^a (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 solution^b 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 Hematoxylin^c 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 agent^d. The mounted slides can be stored at room temperature permanently.

Appendix I

^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)