Amh goat primary antibody

Immunohistochemistry was performed on 4% paraformaldehyde-PBS and Bouin solution-fixed, paraffin-embedded tissues, as previously described26 (link). The Sertoli cells were labeled with an AMH goat primary antibody (1:100; Santa Cruz Biotechnology, CA, USA). Leydig cells were stained with a rabbit anti-cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) antibody (1:250; Sigma Aldrich). A mouse M2A primary antibody (1:100; Abcam, Paris, France) was used for gonocyte immunolabeling. For the AMH and M2A antibodies, antigens were retrieved for 40 min at 80 °C in 10 mM citrate buffer, pH 6.
A NanoZoomer 2.0-RS scanner (Hamamatsu, Tokyo, Japan) was used to capture pictures of the whole slides at 40x magnification. The surface area of 5 to 10 sections randomly selected within the whole explant were calculated with NDP.view software (Hamamatsu). ImageJ software (US National Institutes of Health, Bethesda, MD, USA) was used to perform the stereological cell counting. Germ and Sertoli cells were identified and counted as intra-cordal AMH-negative and AMH-positive cells, respectively.

Sections stained for RNAscope were blocked with PBS-BSA at 10% for 30 min at RT and incubated overnight at 4 °C with primary antibody. Leydig cells were labelled with a rabbit primary antibody directed against CYP11A1 (1:100, Sigma-Aldrich, Ref HPA016436). Germ cells were stained with a LIN28 rabbit primary antibody (1:500, Abcam, Ref Ab46020). Sertoli cells were labelled with an AMH goat primary antibody (1/100, Santa-Cruz, Ref sc6886). Serial frozen sections from MALDI imaging were rehydrated in PBS, submitted to antigen retrieval with citrate buffer as described above, and processed for fluorescent CYP11A1 and AMH double immunostaining. The appropriate AF488 chicken anti-rabbit (Invitrogen, Ref A21441) or AF488 Donkey anti-goat secondary antibody (Invitrogen, Ref A11055) were used at 1:500 for 2 h at RT. PBS with 0.1% of Tween 20 detergent (Sigma-Aldrich) was used for washing steps. Slides were then mounted using Invitrogen Prolong Gold Antifade Mountant with DAPI (Invitrogen, P36935).