Spermatid Perinuclear Ribonucleic Acid: MATERIALS AND METHODS(1)

1 Feb
2013

Mice

Azh mice were obtained from The Jackson Laboratories (Bar Harbor, ME). Hop sterile (hop) mice were provided by Dr. Mary Ann Handel, University of Tennessee, Knoxville, TN. Mice carrying the tw8 and the tw2 haplotypes were from the T/t complex colony at the University of Texas at Austin. All animal experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction. buy ortho tri-cyclen online

Immunocytochemistry

Testes were fixed overnight in Carnoy’s fixative (60% ethanol, 30% CHCl3, 10% glacial acetic acid). The tissues were embedded in paraffin blocks and cut into 6- to 8-^m sections. The sections were deparaffinized with xylene, rehydrated using standard procedures, and incubated with 1: 100 to 1:10 dilutions of affinity-purified primary antibody in PBS for 2 h at 22°C. Antibodies to the SpNr protein have been previously described. Tubulin antibodies (Sigma, St. Louis, MO) were used at a dilution of 1:2000 in PBS. Tissue sections were treated with biotin-conjugated or fluorescently labeled secondary antibodies (Zymed Laboratories, South San Francisco, CA, and Vector Laboratories, Burlingame, CA). Biotinylated antibodies were visualized by light microscopy using a horseradish peroxidase-streptavidin conjugate and aminoethyl carbazole (Zymed and Vector). Fluorescent antibodies were visualized by con-focal microscopy. Confocal images were processed with Adobe Photoshop (Mountain View, CA). Preimmune sera and secondary antibody alone were used as negative controls.

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