Visualizing F-actin in Mouse Testes and GC-1spg Cells

Phalloidin is a natural toxin purified from the mushroom Amanita phalloides to label the F-actin of the cytoskeleton^{58 (link)}. The mice testes were fixed with 4% paraformaldehyde overnight at 4 °C and suspended in Tissue-Tek OCT Compound (SAKURA, USA). The frozen section experiment was carried out. After washing frozen sections with PBS, phalloidin (1:500, Abcam, USA) was incubated for 40 min at 37 °C to show F-actin, and the nuclei were stained with DAPI. For GC-1spg cells, the cells were fixed with 4% PFA for 20 min and washed with PBS after treatment with Si-RhoGDIα and NSC23766 or Y-27632. Then, phalloidin was also incubated for 40 min at 37 °C. The results were captured using the LSM900 confocal laser scanning microscope (Zeiss, Oberkochen, Germany).