Peroxidase affinipure goat anti guinea pig igg h l

C57Bl/6 males (11 weeks of age) were euthanized as per the guidelines of the University of Virginia Institutional Animal Care and Use Committee. Testes were harvested and fixed overnight in Bouin’s fixative (Sigma), followed by embedding in paraffin (Hogarth and Griswold. 2013 (link)). Five-micron-thick sections were deparaffinized in xylene, and rehydrated in a graded series of ethanol baths. Immunohistochemistry was performed on a robotic platform (Autostainer, Dako, Glostrup, Denmark). Endogenous peroxidases were blocked using Peroxidase and Alkaline Phosphatase Blocking Reagent (Dako). No antigen retrieval step was performed. Guinea pig anti-mouse SP-10 primary antibodies (B, C, and D) were used at a 1:1000 dilution. Goat anti-guinea pig-HRP conjugated secondary antibodies (Peroxidase-AffiniPure Goat Anti-Guinea Pig IgG (H+L) Code: 106-035-003, Jackson Immuno Research Laboratories) were used at a 1:200 dilution. The antigen-antibody reaction was assessed by incubation with 3,3’-diaminobenzidinetetrahydrochloride (DAB+) chromogen (Dako), as per the manufacturer’s instructions. All the slides were counterstained with hematoxylin, and were then dehydrated, cleared, and mounted for assessment and imaging. Periodic acid-Schiff histology was performed using a PAS kit (Sigma-Aldrich), following the instructions provided by the manufacturer.

Decapsulated testes from C57Bl/6 males (11 weeks of age) were snap frozen in liquid nitrogen before transferring to −80°C. Cauda epididymides were carefully dissected out, teased, and suspended in sterile phosphate-buffered saline (PBS) for 15 min at 37°C to release sperm. Sperm were collected without letting the pipet tip touch the minced tissue at the bottom of the tube, counted, and pelleted at 6000 rpm at ~4°C. Protein extraction from the frozen testis and sperm samples was performed by brief homogenization followed by extraction with RIPA buffer. Testis and sperm protein extracts as well as recombinant mouse SP-10 protein used for immunization of guinea pigs were subjected to SDS-PAGE, followed by electroblotting onto nitrocellulose membrane (Laemmli. 1970 (link); Towbin and Gordon. 1984 (link)). The blots were incubated with guinea pig anti-SP10 antibodies B, C, and D or preimmune sera at a 1:5000 dilution. Goat anti-guinea pig HRP-conjugated secondary antibodies (Peroxidase-AffiniPure Goat Anti-Guinea Pig IgG (H+L) Code: 106-035-003, Jackson Immuno Research Laboratories) were used at a 1:7000 dilution. Immunoreactivity was visualized by chemiluninescence (Amersham Pharmacia, NJ).