ICC in vitro Sample Clauses

ICC in vitro. Immunocytochemistry (ICC) was performed on cultured structural cells of various types. Cells were grown as described in the cell culture section 2.1 and seeded at 20,000 cells per chamber in four-chamber slides (VWR International). The cells were allowed to reach confluence after which they were gently washed in PBS and then fixed for 10 minutes in 4 % PFA then washed in PBS twice. The chamber slides were stained with the primary layer, mouse monoclonal C3aR (1:50; Abcam) and mouse monoclonal C5aR (1: 40; Abcam) diluted in 5 % normal human serum in PBS. Isotype-matched control antibodies were used as a negative control in the experiments. Incubation was overnight at room temperature. The slides were washed twice in PBS for 5 minutes each time and then treated with the second layer rabbit anti-mouse (1:30; DakoCytomation) in 5 % serum in PBS for 30 minutes. The slides were washed twice again in PBS for 5 minutes each time, and then treated with the third layer mouse PAP (1:50; Xxxxxxx ImmunoResearch Laboratories) for 30 minutes. The PBS washing step was repeated again after which the sections were developed with DAB (Sigma) in the dark for 10-20 minutes; washed with water and then counterstained with Xxxxx’x Haematoxylin (Sigma-Xxxxxxx) and mounted using glycergel. The cells were viewed and photographed with a Zeiss Axioskop 2 microscope equipped with appropriate filters.
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