For histology study, mouse testes were fixed in Bouin's fixative at 4°C overnight for staining with hematoxylin and eosin as described previously (Wang et al., 2017 (link)). For IHF, testes were fixed with 4% paraformaldehyde in PBS at 4°C overnight and embedded in paraffin. The 4-μm-thick testicular sections were incubated with primary antibodies and washed three times with PBS containing 0.5% Tween 20. Nuclei were stained in 0.5 μg/mL DAPI after blotting with FITC- or TRITC-conjugated second antibodies. Slides were imaged under a fluorescent microscope (Leica, DM400BLED368424) or a Lecia TCS/SP5 confocal microscope and processed with Image-Pro Plus. Antibodies and fluorescent probes used in this study: PLZF (Santa Cruz Biotechnology, SC-28319), DDX4 (Abcam, ab13840), SYCP3 (Abcam, ab15093), PCNA (Abcam, ab29), Acrosin/ACR (Atlas Antibodies, HPA048687), rhodamine-labeled PNA (Vector Laboratories, RL-1072), Alexa Fluor 488- or TRITC-conjugated anti-mouse, or anti-rabbit secondary antibodies (Jackson ImmunoResearch).
Rhodamine labeled pna
Manufactured by Vector Laboratories
Rhodamine-labeled PNA is a fluorescently labeled peptide nucleic acid (PNA) probe. PNAs are synthetic DNA/RNA analogs with a neutral peptide backbone. The rhodamine fluorescent label allows for the detection and visualization of the PNA probe.
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Lab products found in correlation
Image pro plus, by Leica (1 mentions)
Rl 1072, by Vector Laboratories (1 mentions)
Sc 28319, by Abcam (1 mentions)
Dm400bled368424, by Leica (1 mentions)
Ab15093, by Abcam (1 mentions)
Ab13840, by Abcam (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
2 protocols using rhodamine labeled pna
1
Histological analysis of mouse testes
2
Immunostaining of Testicular Germ Cells
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Testes were fixed in 4% paraformaldehyde for 2 h at 4C before embedding in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan) for cryosectioning. Then sections of 4 m thickness were prepared and slides were incubated in 0.1% Triton X-100. For staining of GS cells, cells were dissociated using trypsin, and single cell suspensions were concentrated on glass slides by centrifugation using a Cytospin 4 unit (Thermo Elecron Corp, Cheshire, UK). The slides were incubated in 0.01% Triton-X for 15 min for permeabilization. After immersion of slides in the blocking buffer [0.1% Tween 20, 1% bovine serum albumin (BSA) and 10% normal donkey serum in phosphatebuffered saline (PBS)], samples were incubated with indicated antibodies or rhodaminelabeled PNA (Vector Laboratories, Burlingame, CA). OGG1 levels were quantified using the MetaMorph software (Molecular Devices, Sunnyvale, CA). Cells were counterstained with Hoechst 33342 (Sigma, St. Louis, MO). The antibodies used are listed in Supplemental Table S2.
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