The chromosome spreads of spermatocytes were washed with PBS for 3 times and blocked with 5% bovine serum albumin (AP0027, Amresco, Solon, OH). Primary antibodies were incubated at 4 °C overnight, followed by incubation with the secondary antibodies. The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). The images were taken immediately using an LSM 780 microscope (Zeiss, Oberkochen, Germany) or a TCS SP8 microscope (Leica, Wetzlar, Germany). 5-μm sections mounted on glass slides were first deparaffinized and then boiled for 15 min in sodium citrate buffer for antigen retrieval. After washing with PBS, sections were blocked and followed by antibody incubation as described above. To detect apoptotic cells in testis, we used the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay kit (In Situ Cell Death Detection Kit; Roche, 11684795910) according to the manufacturer’s instructions. [53 (link)] Briefly, sections of the testes were deparaffinized and boiled for 15 min in sodium citrate buffer for antigen retrieval. After treated with H2O2 for 10 min at room temperature and sodium citrate for 2 min on ice, the slides were rinsed twice with PBS, the TUNEL reaction mixture was added and incubated in a humidified atmosphere for 60 min at 37 °C in the dark, following by immunofluorescence staining as detailed above.
Protocol detail
Immunofluorescence Analysis of Spermatocytes
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Antibodies
Antibody
Antigen
Apoptotic
Assay
Atmosphere
Bovine serum albumin
Buffer
Cell death
Cells
Chromosome
Deoxynucleotidyl transferase
Dutp
H2o2
Immunofluorescence
Microscope
Nuclei
Sodium citrate
Solon
Spermatocytes
Testes
Transferase
Corresponding Organization :
Other organizations : Institute of Zoology, Chinese Academy of Sciences, University of Gothenburg, Shandong Provincial Hospital, Shandong University, Agency for Science, Technology and Research, Institute of Molecular and Cell Biology, University of Chinese Academy of Sciences
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Variable analysis
independent variables
- Antibody incubation at 4 °C overnight
- TUNEL reaction at 37 °C for 60 min
- Chromosome spreads of spermatocytes
- Apoptotic cells in testis
- PBS washing of chromosome spreads
- Blocking with 5% bovine serum albumin
- Staining with DAPI
- Deparaffinization and antigen retrieval of tissue sections
- Blocking and antibody incubation for tissue sections
- H2O2 and sodium citrate treatment for TUNEL assay
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