EuroMAbNet: European Monoclonal Antibodies Network

Protocols

Immunocytochemistry (ICC) in frozen tissues or cytospins preparations

Procedure

1. Primary Antibody: incubate the frozen tissue sections or cytospins preparations with 100μl of primary antibody at the appropriate dilution for 30 min at room temperature.
2. Wash the slides in 300 ml PBS for 5 min.
3. Repeat the wash in fresh PBS.
4. Secondary Antibody: incubate the sections with 100μl of secondary antibody^a (dilution 1:100 anti-mouse and anti-rabbit / 1:200 anti-rat) for 30 min at room temperature.
5. Rinse the slides in 300 ml of PBS twice as above.
6. 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 minutes until the desired color intensity is reached.
7. Rinse the slides in 300 ml PBS twice as above.
8. Counterstain the slides by immersing them in Hematoxylin^c for 3-4 min.
9. Rinse the slides in running tap water for 3 min.
10. Repeat washing procedure twice.
11. 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)

^a Polyclonal goat anti-rabbit HRP secondary antibody (Dako P0448)

^a Polyclonal rabbit anti-rat HRP secondary antibody (abcam ab6734)

^b Liquid DAB+Chromogen System (Dako K3468) 13,2μl + Hydrogen peroxide solution 3% (Millipore 88597) 11μl + Imidazole 2.5% pH7.5 (1.04716.0250 Millipore) 20μl in 1ml of PBS 1X

^c Carazzi’s Hematoxylin solution DC (Panreac 255298.1212)

^d Aquatex aqueous mounting agent (Merck 1.08562.0050)