After extensive washing with PBS, cells were incubated for 60 min at 37°C with fluorescein iso-thiocyanate (FITC)-conjugated anti-rabbit IgG antibody (1/ 400 dilution in 1% milk in PBS; Sigma) or with biotiny-lated anti-rabbit IgG (1:1000 dilution; Amersham) followed by streptavidin-Cy3 conjugate (1:400 dilution; Sigma). After being rinsed with PBS, preparations were mounted in PBS: glycerol (1:1) containing 5% 1,4-diazabicyclo octane (DABCO; Sigma).
The specificity of scinderin was tested in the bovine adrenals used as a positive control. Moreover, we carried out immunolabeling of bovine testis using another antiserum as an additional positive control, antiserum #7, which is a polyclonal antibody raised against native scinderin and which did not cross-react with gelsolin. For negative controls, we used the primary or the secondary antibody alone. Preimmune serum was also used. In addition, to ascertain further the specificity of scinderin immunolabeling in testicular tissue sections, we performed immunolabeling of paraffin sections of bovine testes with scinderin antiserum #6 preadsorbed with bovine adrenal medulla supernatant.
The protein content of the samples was measured by the Bradford dye binding assay (Bio Rad, ON, Canada).