Publication protocol
The testes were decapsulated, and the seminiferous tubules were carefully separated with microdissection forceps under an Olympus SZX10 stereomicroscope (Olympus, Tokyo, Japan). Several intact seminiferous tubules were fixed in Bouin at 4°C for 2–4 h and then incubated in 100 mmol l−1 glycine at room temperature for 30 min. The samples were then dehydrated and rehydrated in a series of graded methanol (25%, 50%, 75%, and 100%). The tubules were then blocked in 0.3% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and 5% donkey serum (Jackson Immunoresearch Laboratories, Lancaster, PA, USA) in PBS containing 0.05% triton X-100 (Sigma-Aldrich) overnight at 4°C. Then, the seminiferous tubules were incubated with primary antibodies, including rabbit anti-PLZF (1:200 dilution; SC-22839, Santa Cruz), goat anti-PLZF (1:50 dilution; AF2944, R&D Systems), goat anti-GFRA1 (1:50 dilution; AF560, R&D Systems), rabbit anti-TEX14 (1:100 dilution; ab41733, Abcam, Cambridge, UK), and goat anti-c-KIT (1:50 dilution; AF332, R&D Systems) for 3 days at 4°C. After the incubation, samples were washed 5 times with PBS containing 0.05% triton X-100 for 30 min. For whole-mount IHC, the seminiferous tubules were incubated with 3% H2O2 for 30 min and then incubated with biotinylated-linked secondary antibody and streptavidin-conjugated horseradish peroxidase (Maixin Biotech). Samples were visualized using DAB (ZSGB-BIO), counterstained with Harris hematoxylin (BASO, Zhuhai, China), mounted with aqueous mounting media (CW0137, CWBIO, Beijing, China), and imaged with a Sunny RX50 microscope (Sunny). For whole-mount IF, the primary antibodies were detected using donkey anti-goat conjugated to Alexa Fluor 594 or donkey anti-rabbit conjugated to Alexa Fluor 488 secondary antibodies (A21203 or A21206; Thermo Fisher Scientific, Waltham, CA, USA) and counterstained with DAPI (62248, Thermo Fisher Scientific). Images were obtained with a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany). For whole-mount hematoxylin counterstaining histology, several 2–4 cm long seminiferous tubules were fixed in Bouin for 2 h at room temperature, dehydrated and rehydrated in a series of graded methanol, immersed in Harris hematoxylin (BASO) for 2 min, and differentiated in 0.1% acid ethanol for 30 s. The samples were then washed in tap water for 5 min, mounted with aqueous mounting media (CWBIO), and imaged with a Sunny RX50 microscope.
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Manufacturer protocol
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