Tcs sp2 multiphoton confocal laser scanning microscope tcs mp

Aliquots of the sperm capacitated in the presence of 20 μg/ml of rHamOVGP1 for 0, 1, and 3 h, respectively, were used to examine the ability of rHamOVGP1 to bind to sperm cell surfaces. Following incubation as described above, aliquots of 20 μl of sperm were smeared onto superfrost plus microscope slides, air-dried, and fixed for 15 min in 4% paraformaldehyde. The fixed sperm samples were then labeled with a monoclonal antibody against HamOVGP1 (1 μg/ml) or with the same buffer but in the absence of the primary antibody followed by incubation in a solution of goat anti-mouse IgG-FITC at a final concentration of 2 μg/ml. After washing, the samples were mounted with 1% DABCO (1,4-diazobicyclo-[2,2,2]-octane) in 90% glycerol/PBS and then viewed on a Leica TCS-SP2 Multiphoton Confocal Laser Scanning Microscope (TCS-MP, Heidelberg, Germany).

A total of 48 oocytes from mature ovarian follicles were isolated and incubated in TALP-PVA medium in the absence (24 oocytes) or presence (24 oocytes) of immunopurified rHamOVGP1 at a concentration of 20 μg/ml for 3 h at 37°C in 5% CO₂. After incubation, oocytes were washed three times with TL-HEPES buffer, fixed for 30 min in a fixative containing 2% glutaraldehyde and 2% formaldehyde in PBS, blocked with 5% normal goat serum (NGS) in PBS for 1 h prior to incubation in primary antibody (mouse monoclonal anti-hamster OVGP1 antibody diluted in 1% NGS in PBS at a final concentration of 1 μg/ml) overnight at 4°C. Following incubation, oocytes were washed three times for 5 min each in PBS containing 1% NGS. Oocytes were then incubated in fluorescent (FITC)-conjugated goat anti-mouse IgG at a final concentration of 1μg/ml for 1 h in the dark at room temperature. Subsequently, oocytes were washed as above and air-dried, followed by mounting using 1% DABCO in 90% glycerol/PBS. Fluorescent microscopy was performed using a Leica TCS-SP2 Multiphoton Confocal Laser Scanning Microscope (TCS-MP, Heidelberg, Germany).

Capacitated sperm (1 x 10⁵ cells) prepared as described above were smeared onto the surface of superfrost plus microscope slides, air dried, and fixed for 15 min in 4% paraformaldehyde, rinsed in PBS and then permeabilized with 0.1% Triton in PBS at 4°C overnight. In addition, samples of demembranated sperm prepared as described above were smeared onto slides, air-dried, fixed with cold (-20°C) 100% methanol for 1 min, and air-dried. Slides were then washed with DPBS containing 1% normal goat serum (NGS) and blocked with 5% NGS in DPBS for 1 h. Mouse monoclonal anti-phosphotyrosine antibody Clone 4G10 (diluted in blocking solution at a final concentration of 1 μg/ml) was applied to the slides for immunolabeling for 2 h at room temperature. Slides were then washed and incubated with goat anti-mouse IgG-FITC at a final concentration of 1 μg/ml for 1 h in the dark at room temperature. Subsequently, the slides were washed three times with DPBS containing 1% NGS and air-dried, followed by mounting with 1% DABCO in 90% glycerol/PBS. Fluorescent microscopy was performed using a Leica TCS-SP2 Multiphoton Confocal Laser Scanning Microscope (TCS-MP, Heidelberg, Germany).