Whole-mount Immunofluorescence of Seminiferous Tubules

Whole-mount immunofluorescence of seminiferous tubules was performed as described (Carrieri et al., 2017 (link)). For the expression analysis of TAG-NANOS2 and GFRα1, co-staining of tubules was done with two primary antibodies: α-GFP, 1:500 (GFP-1010, Aves), α-GFRα1, 1:50 (GT15004, Neuromics). For the expression analysis of TAG-NANOS2 and c-KIT, co-staining was done with: α-GFP, 1:200 (A-11122, ThermoFisher) and α-C-kit, 1:250 (AF1356-SP, R&D Systems). For the expression analysis of TAG-NANOS2 and PLZF: α-GFP, 1:500 (GFP-1010, Aves), α-PLZF, 1:100 (sc22839, Santa Cruz Biotechnology). For the expression analysis of TAG-NANOS2 and Dcp1a: α-GFP, 1:500 (GFP-1010, Aves), α-Dcp1a 1:500 (WH0055802M6, Sigma-Aldrich). Secondary antibodies used were all Invitrogen: goat anti-Chicken, Alexa Fluor 488 (A-11039); donkey anti-Rabbit, Alexa Fluor 488 (A-21206); donkey anti-goat, Alexa Fluor 568 (A-11057). Images were acquired by using a Confocal microscope, by taking Z-stacks, and with Z-stepsize set at 0.34 μm. Images were analyzed with Fiji ImageJ. A_s and A_pr spermatogonia were distinguished by using the 25 μm topographical criteria (Huckins, 1971 (link)). If the internuclear distance between two spermatogonia was over 25 μm, cells were assigned to the A_s category. On the contrary, spermatogonia whose internuclear distance was smaller than 25 μm, were considered to belong to the same chain.