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).
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.