Anti 11β hsd1

Perfused testes were assembled in five samples per time point in a tissue array and paraffin embedded and 6-μm thick cross sections were cut, mounted, and dewaxed. The immunohistochemical staining for Leydig cell specific protein CYP11A1, 3β-HSD1 and 11β-HSD1 was performed according to the previous method.16 (link)17 (link)18 (link) In brief, the sections were incubated in primary antibody polyclonal rabbit anti-CYP11A1 (RDI Research Diagnostics, Inc., Flanders, NJ, USA; dilution 1:1000), anti-3β-HSD (Abcam, CA, USA, dilution1:1000) and anti-11β-HSD1 (inhouse made, dilution 1:1000), at 4°C overnight. The following morning the sections were washed in Tris-buffer for 5 min; incubated in secondary goat anti-rabbit antibody (Santa Cruz Biotech, CA, USA) 1:1000 diluted in Tris-buffer supplemented for 30 min; washed in Tris-buffer for 5 min and incubated with DAB/H₂O₂ solution for 10 min. They were then washed for 5 min in distilled H₂O and subsequently dehydrated in serial isopropyl alcohol baths and cover-slipped. In control experiments, sections were incubated with nonimmune rabbit IgG (CYP11A1, 3β-HSD1 and 11β-HSD1).

Testes were homogenized and boiled in equal volumes of sample loading buffer, a Tris-Cl buffer (pH 6.8), containing 20% glycerol, 5% sodium dodecyl sulfate, 3.1% dithiothreitol, and 0.001% bromophenol blue. Homogenized samples (50 μg protein) were electrophoresed on 10% polyacrylamide gels containing sodium dodecyl sulfate. Proteins were electrophoretically transferred onto nitrocellulose membranes, and after 1 h exposure to 5% nonfat milk to block nonspecific binding, the membranes were incubated with a rabbit polyclonal anti-11β-HSD1 (custom antibody, dilution 1:1000),18 (link) 3β-HSD1(Abcam, San Francisco, CA, USA, dilution 1:2000), CYP11A1 and FABP3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA dilution 1:1000), CYP11A1 and steroidogenic acute regulatory protein (STAR) (kindly donated by Dr. D.M. Stocco, Texas Tech Health Sciences Center, Lubbock, TX, USA, dilution 1:1000 ~ 5000), and actin β (ACTB, Cell signaling technology, Danvers, MA, USA, dilution 1:1000) antibodies. The membranes were then washed and incubated with a 1:5000 dilution of goat anti-rabbit antiserum that was conjugated to horseradish peroxidase. The washing step was repeated, and immunoreactive bands were visualized by chemiluminescence using a kit (ECL, Amersham, Arlington Heights, IL, USA).