Plzf sc 28319

Primary antibodies against Shp2 (sc-280), Wt1 (sc-7385), PLZF (sc-28319), Cx43 (sc-9095), UPA (sc-14019), TPA (sc-81693), ABP (sc-32891), AR (sc-816), PDGFA (sc-128), DMRT1 (sc-10485), ERK1/2 (sc-292838), Akt1/2/3 (H-136, sc-8312) and AMH (sc-6885) were obtained from Santa Cruz Biotechnology, Dallas, Texas, USA. Antibodies for phospho-Akt (Ser473, 4060), phospho-ERK1/2 (Thr202/Thr204, 4370) and PH3 (9701s) were purchased from Cell Signaling Technology, Danvers, Massachusetts, USA. Antibodies against SCF (ab64677), Stra8 (ab49405), SCP3 (ab97672), Claudin11 (ab53041), FSHR (ab35309), GDNF (ab18956) and pem (ab31922) were purchased from Abcam, Cambridge, Massachusetts, USA. Other antibodies included JAMA (36–1700, Invitrogen, Grand Island, New York, USA) and BMP4 (SAB2700755, Sigma Chemical Co., St. Louis, Missouri, USA). All chemical reagents were purchased from Sigma-Aldrich and Solarbio (Shanghai, China).

Cells on Matrigel (BD Bioscience) coated cover slides were fixed in 4% paraformaldehyde (Sangon Biotech, Shanghai, China) at 4 °C overnight, and immunofluorescence (IF) assays were performed according to published protocols^{55 (link)}. The following antibodies were used: PLZF (SC-28319, Santa Cruz Biotech, Dallas, TX, USA), STRA8 (ab49602, Abcam, Cambridge, MA, USA), SYCP3 (ab15093, Abcam), Alexa Fluor 488- or TRITC-conjugated anti-mouse and anti-rabbit secondary antibodies (115-545-146, 115-025-146; 111-545-144, and 111-025-144, Jackson ImmunoResearch, West Grove, PA, USA). Nuclei were stained with 0.5 µg/mL DAPI before being visualized using an Olympus microscope BX53. Images were processed with the Image-J software. For histology studies, testes were fixed in Bouin’s fixative at 4 °C overnight for staining with hematoxylin and eosin, as previously described^{56 (link)}. For immunohistofluorescence (IHF), testes were fixed with 4% paraformaldehyde in PBS at 4 °C overnight and embedded in paraffin. Testis sections were stained with an ACR antibody (HPA048687, Atlas Antibodies, Sweden), followed by staining with an Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch) and Rhodamine-labeled Peanut Agglutinin/PNA (RL-1072, Vector Laboratories, USA). Images were collected using a fluorescent microscope (Leica, DM400BLED368424).