Local Regulation of Macrophage Subsets in the Adult Rat Testis: MATERIALS AND METHODS(6)

Western BlotAnalysis

Residual samples of testicular IF from each experimental group were pooled and were subjected to Western blot analysis as previous described, with some modifications. The pooled samples were diluted 1:1 with sample buffer (0.125 M Tris, 4% SDS, 20% glycerol, 2.5% (3-mercapto-ethanol, 0.02% bromophenol blue, pH 6.8). The equivalent of 6-10 ^l of IF from each experimental group was run on a 15% SDS-polyacrylamide gel and transferred onto Im-mobilon P membrane (Millipore, Bedford, MA) in a 20 mM Tris, 150 mM glycine buffer containing 20% methanol at 0.5 mA/cm2 overnight. Excess binding sites on the membrane were blocked with high-salt (350 mM NaCl) Tris-buffered saline, pH 7.4 (TBS), containing 5% (w:v) nonfat dry milk powder, 2% normal goat serum, and 0.2% Tween 20, for 1 h. The membrane was probed (1 h) with rabbit anti-MIF polyclonal antibody diluted 1:1000 in the blocking buffer, followed by a donkey anti-rabbit IgG-peroxidase (Amersham, Arlington Heights, IL) diluted 1:10 000 in the blocking buffer (1 h). The blot was washed five times (5 min each) with TBS, 1% (v:v) Tween 20. After a final rinse with TBS, MIF-reactive bands were revealed using an enhanced chemiluminescence system (Amersham).

Statistical Analysis

Comparisons between experimental groups were made by two-way ANOVA after appropriate transformation to normalize data and equalize variance where necessary, or by Kruskal-Wallis ANOVA on ranks, in conjunction with Student-Newman-Keuls multiple range test. Student’s t-test was used for comparing data from two groups. Correlations were determined by Pearson’s product-moment correlation analysis. All statistical analyses were performed using Sig-mastat version 1.0 software (Jandel Corp., San Rafael, CA). All values are mean ± SEM (n = 6 animals per group) unless otherwise indicated.

This entry was posted in Adult Rat Testis and tagged Adult Rat Testis, Inhibitory Factor, Macrophage, Seminiferous Tubules, Testosterone.