Tritc labelled goat anti mouse igg

Oocytes were washed three times with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde in PBS at 4 °C for 1 h. Fixed cells were washed 3 times with PBST (PBS supplemented with 0.1% Tween 20) and incubated in 0.1% Triton X-100 in PBS at room temperature for 30 min. After washing with PBST, oocytes were blocked with 5% bovine serum albumin in PBS at room temperature for 1 h and transferred into diluted media containing a rabbit anti-Mfn2 antibody (1:50, a kind gift of Professor Chen Kuang-Hueih), a monoclonal anti-tubulin antibody (1:100), or a rabbit anti-p-p38 antibody (1:100) either at room temperature for 2 h or overnight at 4 °C. Finally, the labelled oocytes were washed with PBST and stained with FITC-labelled goat-anti-rabbit IgG (1:100) and TRITC-labelled goat-anti-mouse IgG (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 2 h. For mitochondrial staining, oocytes were incubated for 30 min at 37 °C in M2 medium supplemented with 200 nM MitoTracker Red. The oocytes were finally stained with Hoechst 333342 after three washes in washing buffer and were mounted on glass slides for immunofluorescence microscopy. Photos were captured using a confocal laser-scanning microscope (Zeiss LSM 780, Oberkochen, Germany).