Freshly released epididymal sperm were used for SIM super-resolution microscopy. Sperm were collected, as described previously, with the following differences. Sperm samples were always prepared onto high precision cover glasses (thickness No. 1.5 H, 170 ± 5 μm, Marienfeld, Germany). Moreover, after the application of the primary and secondary antibodies, sperm were incubated for 5 min with DAPI (0.85 μg/mL, Thermo Scientific, Waltham, MA, USA) and washed 3× in PBS. At the end, sperm were washed 1× in distilled water and air-dried. Dry samples were covered with 90% glycerol with 5% anti-fade N-propyl gallate (Sigma-Aldrich). Multi-colour SIM super-resolution images were obtained by Zeiss Elyra PS.1 inverted microscope at Laboratory of confocal and fluorescent microscopy of Faculty of Science (Charles University, Prague, Czech Republic). An open source software Fiji was used for another image processing.
Anti fade n propyl gallate
Manufactured by Merck Group
Anti-fade N-propyl gallate is a compound used as a mounting medium additive in microscopy. It helps to prevent the fading of fluorescent signals over time. The product maintains the integrity of fluorescent samples during imaging and analysis.
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Lab products found in correlation
2 protocols using anti fade n propyl gallate
1
Super-resolution imaging of epididymal sperm
2
Fluorescent Imaging of Sperm Cells
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Freshly released sperm were used for STED imagery. Sperm were collected the same way as described previously with some differences. Sperm samples were always prepared onto a microcover glass (thickness 0.13-0.17 mm; Hirschmann). Moreover, after application of primary and secondary antibodies, sperm were incubated for 30 min with 2.5 mM PNA lectin (Molecular Probes, L-32458) in PBS. After washing, DAPI (0.85 μg/mL; Thermo Scientific) was added for 5 min and washed three times in PBS. At the end, sperm were washed once in distilled water and air-dried. Dry samples were covered with 90% glycerol with 5% anti-fade N-propyl gallate (Sigma Aldrich). Fluorescent images were collected with a Leica TCS SP8 STED 3X microscope using the software LAS X 64bit package with LAS AF SP8 Dye Finder, 3D visualization, deconvolution and colocalization module (Microscopy Centre, IMG AS, Prague, Czech Republic).
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