Alexa 488 donkey antirabbit

Snap‐frozen tissue biopsies of histologically normal human skin, ileum, and colon were cut at 4 µm and sections were adhered to SuperFrost slides by thaw‐mounting and air‐dried. Sections were stained with rabbit‐antihuman TG2 (custom‐made polyclonal antibody from Pacific Immunology, CA) or with rabbit–antihuman TG3 (PA596450, Thermo Fischer Scientific) followed by detection with Alexa 488 donkey antirabbit (Molecular Probes) antibody. Slides were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and mounted with ProLong Diamond Antifade Mountant (ThermoFisher). Images were acquired using a 20× objective lens Zeiss LSM 880 confocal laser scanning microscope.

Spreads of spermatocytes and immunofluorescence staining were prepared according to the previous references51 (link)52 (link). Briefly, seminiferous tubules were incubated in hypotonic extraction buffer (50 mM Sucrose, 17 mM Sodium citrate, 30 mM Tris (pH 8.2), 2.5 mM DTT, 1 mM PMSF (pH 8.3) and 5 mM EDTA on ice for 20 minutes, minced in 100 mM sucrose, spread on slides and fixed in 1% PFA with 0.1% Triton X-100. Slides were incubated in a humid chamber overnight, dried, and washed in PBS and water containing Photoflo (Kodak, NY, USA). Following blocking in 10% donkey serum and 3% BSA, immunofluorescence staining was performed by incubating with primary antibodies: γH2AX (1:500; abcam) and SYCP3 (1:100; Abcam) overnight at room temperature. Alexa 488 donkey anti-rabbit (1:500, Molecular Probes), Alexa 594 goat anti-mouse (1:200, Molecular Probes) were used as secondary antibodies. Slides were incubated with secondary antibodies at 37 °C for 1 hour in the dark, washed and mounted with Vecta shield cover slips. (Vector Laboratories).

Double immunofluorescence was carried out as described previously [25 (link)]. Briefly, lung sections were mounted on superfrost plus microscope slides (VWR Scientific, Radnor, PA, USA) and deparaffinized in xylene (5 min ×2), rehydrated through a range of aqueous ethyl alcohol solutions in H₂O, and immersed in PBS for 5 min. Antigen retrieval was performed by incubating the sections in 10 mM citrate buffer, pH 6, in a microwave oven for 5 min. The slides were then incubated in a blocking solution (5% normal donkey serum, 0.5% Triton in PBS) for 1 h at room temperature, followed by an overnight incubation at 4 °C with the primary antibody Slc2a1 (1:400) in blocking solution. The next day, the slides were washed with PBS for 10 min ×3 and incubated in Alexa 488 (donkey anti-rabbit, 1:300, green color, Molecular Probes) at room temperature in a dark place for 1 h, followed by washing in PBS for 10 min ×3. For double staining, the sections were blocked again, as described earlier, and incubated in the smooth muscle α-actin antibody (1:15) overnight; the procedure was repeated using the appropriate secondary antibody Alexa 594 (donkey anti-rabbit 1:200, red color, Molecular Probes, Eugene, OR, USA). The sections were examined with a laser scanning confocal fluorescence microscope (MRC 1000, Bio-Rad). The negative controls were run in the absence of primary antibodies.