Alexa fluor 488 conjugated anti rabbit secondary antibody

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).

To determine ERα expression, cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized in PBS containing 0.1% Triton X-100 for 10 minutes, and incubated in PBS containing 1% BSA (Sigma-Aldrich), 2% FBS (Gibco), and 10% normal donkey serum (Jackson ImmunoResearch) for 1 hour. Coverslips were incubated with anti-ERα rabbit monoclonal (1:250; Abcam, ab16660) primary antibody overnight at 4°C. After washing, coverslips were incubated with Alexa Fluor 488–conjugated anti-rabbit secondary antibody (1:1000; Jackson ImmunoResearch, 711-545-152) for 45 minutes at room temperature. Nuclei were stained with DAPI (0.5 μg/mL) and coverslips were mounted with Dako Mounting Medium (Agilent Technologies). Microscopy imaging was performed with a Leica DMi8 microscope at ×40 magnification.