Acrosome Biogenesis in the Hamster: MATERIALS AND METHODS(5)
Coverslips were then rinsed three times in PBS containing 1% goat serum (PBS-GS) and incubated in primary antibody diluted in PBS-GS; controls received equivalent dilutions of preimmune serum or normal ascites fluid. After three washes in PBS-GS, the coverglasses were incubated in PBS-GS containing affinitypurified secondary antibodies of fluorescein isothiocyanate (FITC)-conjugated anti-guinea pig IgG or Cy3-conjugated anti-mouse IgG (KPL Inc. and Jackson Immuno Research, West Grove, PA, respectively). Coverslips were then rinsed several times in PBS, and cells were examined by phase contrast and epifluorescence microscopy. For double-antibody immunostaining, cells were incubated with a mixture of monoclonal and polyclonal primary antibodies and then in a mixture of anti-guinea pig and anti-mouse secondary antibodies conjugated with different fluorochromes. As a control, each secondary antibody was tested individually against both primary antibodies, and no species cross-reactivity was noted.
For post-embedding immunolabeling, cells were fixed on ice with 4% formaldehyde, 0.25% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The samples were then rinsed with phosphate buffer, dehydrated through an ethanol series, and embedded in LR White Resin (Ted Pella Inc., Burlingame, CA). Thin sections were mounted on nickel grids and immunostained as described for immunofluorescence except that gold-conjugated secondary antibodies were used (Amersham Inc., Arlington Heights, IL). After immunolabeling, the sections were washed in PBS, fixed with 1% glutaraldehyde, rinsed with water, and stained with uranyl acetate and lead citrate. buy asthma inhalers
For pre-embedding immunolabeling, spermatozoa were permeabilized and immunolabeled as for immunofluorescence except that gold-conjugated secondary antibodies were used. After labeling, sperm were fixed with 4% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4), postfixed with 1% osmium tetroxide in sodium cacodylate buffer, dehydrated in an ethanol series, equilibrated with propylene oxide, and embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate.