Calcium-Dependent Actin Filament: MATERIALS AND METHODS(5)



The sections were first incubated for 30 min at 37°C with 0.5% skim milk in TBST to block the unspecific binding and then incubated overnight at RT with (1:400) scinderin antiserum #6 and then for 40 min with (1:1000) biotinylated anti-rabbit IgG (Amersham Bio/Can Scientific, Mississauga, ON, Canada) followed by (1:200) horseradish-peroxi-dase (HRP)-conjugated streptavidin (Amersham). They were washed in TBST and incubated for 10 min at RT in 0.01% H2O2, 0.05% diaminobenzidine tetrachloride (DAB), and 10 mM imidazole in TBS (pH 7.7). The sections were counterstained with methylene blue dye and mounted with Permount (Fisher Scientific Co., Pittsburgh, PA). The recordings of the stage-dependent distribution of scinderin in the adult bovine testis were made using the identification method of the twelve stages of the cycle of the seminiferous epithelium proposed by Berndtson and Desjardins.

Fluorescence Microscopy

Spermatozoa were spotted on poly-L-lysine-coated glass coverslips and air-dried. Spermatozoa were fixed-permea-bilized by incubating the coverslips in (-20°C) methanol for 10 min. Preparations were washed with PBS and incubated with 3% skim milk in PBS for 60 min at RT to block nonspecific binding. Next, cells were incubated with scinderin antiserum #6 (1/80 dilution in 1% milk in PBS) for 60 min at 37°C.


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