Sc 32251

Ovaries of superovulated 3.5- to 4-week-old Abhd2^+/+ and Abhd2^–/– mice were fixed overnight at 4°C in 4% PFA, and the ovary of a 1-week-old Abhd2^+/+ female was fixed for 4 h after which they went through a sucrose gradient (10−20−30% sucrose/PBS) and were frozen in OCT. To detect localization of ABHD2 in the ovary, 8-μm sections were placed on charged slides and immediately used for staining, according to standard procedures. In brief, for antigen retrieval, the sections were incubated in 1% SDS solution for 5 min, blocked in 5% BSA for 1 h at room temperature, and incubated overnight at 4°C with rabbit polyclonal anti-ABHD2 (1:300) and mouse monoclonal anti-α-actin (1:500, sc-32251, Santa Cruz Biotechnology). The antibody–antigen complexes were visualized by incubation for 1 h at room temperature with 1:2,000 Alexa Fluor 488-conjugated goat anti-rabbit (Molecular Probes A11008/Jackson ImmunoResearch, 111-485-144) and Cy5-conjugated donkey anti-mouse (Jackson ImmunoResearch, 715-175-150) or anti-rabbit (Jackson ImmunoResearch, 711-015-152) antibodies. The sections were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen, P36935) and imaged using confocal laser scanning microscopy (Olympus Fluoview FV1000).

Tissue was collected from sexually mature mice (>8 weeks old).
Mlh3^−/− mice (The Jackson Laboratory; #018845) were bred at the Washington University in Saint Louis and processed as previously described (Jung et al., 2019 (link)). Mice of the Crispy line were bred by the Ahituv laboratory at the University of California, San Francisco, and fixed testes were received in 70% ethanol. Wild-type C57BL/6J (The Jackson Laboratory; #000664) were bred in-house at the Small Laboratory Animal Unit (ONPRC/OHSU). All animal experiments were performed in compliance with the regulations of the respective host institutions.
To capture subtle image differences resulting from experimental variables, the samples used to train the neural network were processed under different conditions: (i) fixation method—perfusion or immersion; (ii) fixative type—4% paraformaldehyde (PFA) or modified Davidson’s fixative and (iii) assay—IF with or without Tyramide signal amplification and RNA fluorescence in situ hybridization followed by immunofluorescence. The boundaries of the seminiferous tubules were detected with anti-ACTA2 (1:100, Santa Cruz, sc-32251), the staging of tubules was performed with anti-ACRV1 (1:200, Proteintech, 14040-1-AP) and Sertoli cell nuclei marked using anti-SOX9 (1:100, Sigma-Aldrich, HPA001758). See the Supplementary Methods for a detailed description of the experiments.

VICs were seeded onto chamber slides pre-coated with collagen I and differentiated into myofibroblasts during 14 days as it was described above. Then the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, P6148) diluted in PBS for five minutes at room temperature, permeabilized in 0.5% Triton X-100/PBS for five minutes and blocked with 10% FBS/PBS at room temperature for 30 minutes. Then the cells were incubated for one hour with primary antibodies: mouse monoclonal anti-α-SMA (sc-32251, Santa-Cruz) and rabbit polyclonal anti-calponin (ab46794, Abcam) and washed with PBS. The respective secondary antibodies Alexa 647-labeled goat anti-mouse IgG2a (A21241, Invitrogen) and Alexa 488-labeled goat anti-rabbit IgG (H + L) (A11008, Invitrogen) were applied for 40 minutes at room temperature. After washes with PBS, the nuclei were stained with Hoechst (33258, Invitrogen). Then cells were washed with PBS and visualized by epi-fluorescence microscopy (Axio Observer Z1, Zeiss).

To induce myofibroblast differentiation MEFs were plated on glass coverslips with the appropriate medium. After serum starvation for 24 h, the cells were preincubated with RGZ or VCE-004.8 for 1 h and stimulated with TGFβ1 (10 ng/ml) for another 24 h. Coverslips were collected, washed with PBS and fixed with 4% paraformaldehyde. After antigen retrieval non-specific binding was reduced by blocking with IHC select blocking reagent (Merck Millipore, MA, USA) at room temperature for 30 min. Coverslips were placed in a humidified chamber and incubated overnight at 4 °C with a primary antibody α-SMA (1:50, sc-32251, Santa Cruz). Then, coverslips were washed three times with 0.1% PBS-Tween 20 and incubated with the secondary antibody goat anti-rat Alexa 488 (Merck Millipore). Finally, coverslips were mounted in VectaShield Mounting Medium with DAPI and analyzed using a Leica DM2500 microscope and a Leica DFC420c camera.

For the characterization of DPCs, α-smooth muscle actin (SMA) is one of the marker genes. To detect the immune-staining, we used primary antibody against SMA (sc-32251, Santa Cruz Biotechnology, Dallas, TX, United States). The seeding condition is identical with that of F-actin in the previous section. Cells were washed by 1X PBS, then fixed with 4% paraformaldehyde solution. After the permeabilized treatment with Triton X-100, we did the blocking with 1% bovine serum albumin in PBS for 10 min. Next, X100 diluted primary antibody was exposed to the cell overnight at 4°C. After the wash with 1X PBS wash, the secondary antibody, Goat anti-Mouse IgG (H + L) with Alexa 488 (Thermo Fisher Scientific) was exposed to the cells with X 200 dilution for 1 h. DAPI (4′,6-diamidino-2-phenylindole) was used as a counterstaining reagent. We detected the intensity of fluorescence images with imageJ software. The intensity was detected from the cytoplasm of 15 randomly selected cells.