After GLIFin-mediated light inactivation of F-actin as described above, cells were washed with PBS three times, fixed, and permeabilized with PBS containing 4% HCHO and 0.1% Triton X-100. After 10 min, the solution was aspirated. The fixed cells were washed with PBS three times and blocked with 1% bovine serum albumin (BSA)/PBS. After 30 min, the blocking solution was aspirated, and the fixed cells were incubated in PBS containing 1% BSA, 0.66% MeOH, Alexa Fluor 647 phalloidin (2 U/ml) (A22287, Thermo Fisher Scientific), anti–α-tubulin antibody conjugated with fluorescein isothiocyanate (FITC) (3 μg/ml; ab64503, Abcam), and 4′,6-diamidino-2-phenylindole (DAPI) (3 μg/ml; D1306, Invitrogen) at ambient temperature for 1 hour. The PBS was aspirated and replaced with fresh PBS. Fluorescence imaging was done at 405/430 to 465 nm for DAPI, 488/510 to 550 nm for FITC (tubulin), and 633/661 to 750 nm for Alexa Fluor 647 (F-actin).
Ab64503
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab64503 is a lab equipment product offered by Abcam. It serves a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
A22287, by Thermo Fisher Scientific (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Ab48394, by Abcam (1 mentions)
Ab150073, by Abcam (1 mentions)
3 protocols using ab64503
1
Multicolor Cytoskeleton Visualization
2
Immunofluorescence Staining of Mouse Oocytes
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The method of immunofluorescence manipulation was referenced in our previous study [21] (link). The mouse IVM oocytes were first rinsed and fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for 30 min, and blocked in 3% BSA in PBS for 2 h at room temperature. Incubation was carried out overnight at 4°C with primary antibody (1∶500, ab64503, Abcam, UK). After rinsing, the embryos were incubated under the same conditions with fluorescein isothiocyanate (FITC)-conjugated secondary antibody for 2 h. The nuclear status of embryos was evaluated by staining with 10 µg/mL of propidium iodide for 10 min. Finally, the embryos were mounted on glass slides and examined with a confocal laser scanning microscope (LSM710 Carl Zeiss, Oberkochen, Germany).
3
Immunofluorescence Analysis of Autophagy in Oocytes
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Oocytes were fixed in PBS-PVA containing 3.7% paraformaldehyde for 30 min and permeabilized by incubation in 0.3% Triton X-100 for 15 min at room temperature. Oocytes were then blocked in PBS-PVA containing 1% BSA for 1 h. Next, oocytes were incubated with primary anti-LC3B antibody (Abcam, Cambridge, MA, United States; #ab48394) or anti-α-tubulin-FITC antibody (1:200; Abcam; #ab64503) overnight at 4°C. After washing three times in PBS-PVA, the oocytes were incubated with a secondary antibody (Abcam; #ab150073, for LC3B staining) for 1 h at room temperature. Then, DNA was stained with 1 μg/mL Hoechst 33342 for 15 min. Finally, the oocytes were mounted onto glass slides and the fluorescence intensities were examined using a confocal laser scanning microscope (Carl Zeiss). Autophagy levels in the embryos were measured by counting the number of LC3B dots.
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