Spermatocyte spreads were performed as previously reported (Garcia-Cruz et al. 2011 (link); Capilla et al. 2014 (link); Marín-Gual et al. 2022 (link)). Immunostaining of meiocytes was performed using the following primary antibodies: rabbit antibody against SYCP3 (#ab15093, Abcam, 1:400 dilution), mouse antibody against MLH1 (#ab14206, Abcam, 1:50 dilution), human calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly and telangiectasia (CREST) serum (a kind gift of M. Fritzler, 1:100 dilution). Fluorochrome-conjugated secondary antibodies were used for detection (all from Jackson ImmunoResearch Laboratories, 1:200 dilution). The immunostaining protocol included antigen retrieval with incubation in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20; pH 6.0) at 95 °C for 15 min and permeabilization steps with PBST (0.05% Triton X-100 in PBS), and overnight incubation for primary antibodies at 4 °C and 1 h for secondary at 37 °C, both in a humidified chamber.
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Meiotic Chromosome Immunostaining Protocol
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Other organizations : Universitat Autònoma de Barcelona, Hospital General de Granollers
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Variable analysis
independent variables
- Spermatocyte spreads were performed as previously reported
- Immunostaining of meiocytes was performed using the following primary antibodies: rabbit antibody against SYCP3, mouse antibody against MLH1, human calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly and telangiectasia (CREST) serum
- Not explicitly mentioned
- Sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20; pH 6.0) at 95 °C for 15 min (for antigen retrieval)
- PBST (0.05% Triton X-100 in PBS) (for permeabilization)
- Overnight incubation for primary antibodies at 4 °C and 1 h for secondary at 37 °C, both in a humidified chamber
- Positive control: Not specified
- Negative control: Not specified
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