Streptavidin peroxidase

IHC was conducted to localize the NR1D2 protein in human testicular tissues. Human testes were dissected into pieces, fixed with 4% PFA, embedded in paraffin, and sectioned at 4 um. To confirm the specific infertility syndrome, sections were stained with haematoxylin and eosin following a standard protocol. Initially, slides with testicular tissue sections were heated in 10 mM sodium citrate buffer (pH 6.0) for 10 min, after deparaffinization in a microwave oven. The sections were then dipped into PBS containing 3% H₂O₂ and 0.1% Triton X-100 to quench endogenous peroxidase activity. After treatment with 10% normal donkey serum (Jackson ImmunoResearch Labs Inc., West Grove, PA, USA) to block non-specific binding signals, the slides were incubated with NR1D2-specific antibody (Proteintech Group, Inc.) overnight at 4 °C and then incubated with a mouse biotinylated secondary antibody (Abcam, Cambridge, MA, USA) for 2 h at room temperature. Immunoreactivity with NR1D2 was visualized using streptavidin-peroxidase and 3,3′-diaminobenzidine (Maixin Bio, Fuzhou, China).

The bones, livers, spleens, intestines, and kidneys from the transplanted mice were obtained at specified points in time and fixed in 10 % paraformaldehyde for 24 h. They were then processed for paraffin embedding. Five-to-seven-micrometer-thick sections were obtained, deparaffinized in xylene (Sinopharm Chemical Reagent Co. Ltd., China), and rehydrated in grades of ethanol. One set of tissue sections was treated with H&E staining and the other set was applied to immunohistochemical staining using anti-human CD45 antibody as described previously. Briefly, endogenous peroxidase was inactivated using 3 % hydrogen peroxide for 30 min. The slides were incubated in goat serum for 30 min to block non-specific binding of the primary antibodies. The sections were stained with rat anti-human CD45 (Abcam) for 30 min at room temperature, followed by biotinylated goat anti-rat IgG for 20 min and streptavidin peroxidase for 20 min (both from Maixin Bio, China). The antibodies were tested for specificity on murine bone marrow to exclude interspecies cross-reactivity. Positive signals were visualized with 3,3′-diaminobenzidine (DAB) chromogens.

For HMGB1 detection, the samples were dewaxed in xylene and dehydrated in a graded ethanol series. Endogenous peroxidase activity was inhibited by incubating the slides with 0.3% H₂O₂ for 5 min, followed by washing thrice with PBS; the sections were incubated with the primary antibodies to HMGB1 (1 : 1000 dilution, ab18256; Abcam, Cambridge, UK) and incubated at 4°C overnight, washed in PBS, and incubated at 37°C for 1 h with biotinylated anti-rabbit/rat IgG (1 : 200; Maixin-Bio, Shanghai, China) according to manufacturer's instructions. The tissue was incubated with Streptavidin Peroxidase (Maixin-Bio) reagents at 37°C for 30 min, stained with freshly prepared DAB (Maixin-Bio). Morphometric quantification of the stained sections was performed with a customized digital image analysis system (IMAGE-Pro plus 4.5). Analysis of the kidney and capturing images was performed.