Alexa fluor 488 or 594 conjugated secondary antibodies

Testes were fixed in Bouin's buffer or 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned. The sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). For immunofluorescence analysis, the sections were boiled in 10 mm sodium citrate buffer (pH 6.0) for 15 min, cooled to 25 °C, washed, blocked with 10% donkey serum for 60 min, and incubated with primary antibodies overnight at 4 °C. Then, the slides were washed and incubated with Alexa Fluor 488‐ or 594‐conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) for 1 h. The slides were mounted with DAPI (Molecular Probes) and imaged under a confocal microscope equipped with a camera (Olympus).
For immunohistochemical analyses, incubation with primary antibodies was performed using biotin‐labeled secondary antibodies. The antibodies were further developed using Vectastain ABC kit and 3,3‐diaminobenzidine peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA). The slides were then counterstained with hematoxylin and mounted.

VNOs embedded in OCT medium were sliced in 16 μm thick sections in a cryostat apparatus (Leica CM 3050S). Slices were incubated in blocking solution containing 0.5% Triton X-100 and 5% normal horse serum in 0.1 M TBS for 2 h at room temperature (RT). Primary antibody incubation was performed overnight (o/n) at 4 °C with anti-OMP (Wako goat polyclonal; 1:2000), anti-PCNA (Sigma, rabbit monoclonal; 1:2000), and anti-Sox2 (R&D Systems, goat polyclonal; 1:300). For PCNA immunostaining, slices were incubated in 10 mM citrate buffer (100 °C; pH, 6.0) for 5 min prior staining. Sections were incubated with Alexa Fluor 488- or 594-conjugated secondary antibodies (Jackson Laboratories, 1:500) for 2 h at RT. Hoechst (Sigma, 1:10,000) was added during 5 min after the secondary antibody incubation for nuclei visualization. Imaging was performed using a vertical confocal microscope Leica SPEII. Final images were assembled in Adobe Illustrator.

Cells cultured on Lab-Tek chamber slides were fixed with paraformaldehyde and rinsed in PBS. Cells were incubated with anti-Crk or anti-DOCK180 antibodies (dilution 1:100) overnight and then with Alexa Fluor 488- or 594-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature for 2 hr. Cells were counterstained with DAPI for 15 min. Fluorescent images were taken with the confocal microscope.

For the immunostaining of cells, cells were first seeded onto gelatin‐pre‐treated slides and were then fixed with 4% PFA. After blocking with 10% horse serum in PBS plus 3%, the slides were incubated with the appropriate anti‐NUDT21 and anti‐EZH2 primary antibodies and Alexa Fluor 488‐ or 594‐conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, USA). Cell nuclei were detected with DAPI (Invitrogen) then imaged under a confocal microscope (Tcs‐sp5‐II; Leica, Wetzlar, Germany).
For the immunohistochemical detection of proteins in tumour tissue, placental tissue samples were fixed with 4% PFA, dehydrated and then embedded in paraffin. The paraffin‐embedded tissues were then sectioned using a microtome (RM2245; Leica), and the slide‐mounted sections were deparaffinized and rehydrated. Antigens were retrieved by microwaving in sodium citrate buffer (10 mmol/L sodium citrate acid, 0.05% Tween 20, pH 6.0) for 2 minutes. After blocking with 3% H₂O₂, followed by normal human serum for 1 hour, the sections were subjected to immunohistochemical staining using the appropriate anti‐NUDT21 and anti‐EZH2 primary antibodies and HRP‐conjugated secondary antibodies. The signals were then detected with DAB substrate (ZSGB‐BIO, Beijing, China), and the sections were counterstained with hematoxylin.

The 6-μm paraffin sections were deparaffinized, rehydrated, antigen-retrieved, blocked, and then incubated with primary antibodies overnight at 4 °C. The antibodies included anti-KLF4, anti-SP7, anti-BGLAP (ab93876; Abcam), and anti-ALPL (AF2910; R&D Systems, Minneapolis, MN, USA). The sections were then incubated with Alexa Fluor 488- or 594-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA, USA). Finally, the stained sections were mounted with DAPI.

Rats were euthanized and intracardially perfused first with phosphate‐buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were then post‐fixed overnight and cryoprotected with 30% sucrose at 4°C for 24–48 hr. Twenty ųm thickness sections were cut by Leica Cryostat Microsystems CM 1900‐3‐1 and left to dry for 10 minutes at Room Temperature (RT). Immunostaining was performed as previously described (Ahmed et al., 2017). Briefly, cryosections were washed three times in PBS, then blocked for 1 hour at RT with blocking solution (3% BSA and 0.2% Triton X‐100 in PBS). Anti‐NeuN antibody (ab177487; rabbit; 1:1,000; Abcam) and Recombinant Anti‐Amyloid Precursor Protein antibody (ab32136; rabbit; 1:200; Abcam) were then applied for 24 hr at 4°C followed by three rinses with PBST buffer (0.2% Triton X‐100 in PBS). Tissues were subsequently incubated with Alexa Fluor 594‐ or 488‐conjugated secondary antibodies (1:500; from Jackson ImmunoResearch, West Grove, PA) for 3 hr at RT. Hoechst 33,342 (Hst) was used to counterstain the nuclei. After triple washing with PBST buffer, the samples were mounted and cover‐slipped with anti‐fading medium (2.5% PVA‐DAPCO) and examined by Olympus “BX60F5” Fluorescence Microscope.