Alexa488 coupled anti mouse

Embryo fixation and staining for indirect immunofluorescence was performed mostly as described (Gönczy et al., 1999 (link)), using 1:200 mouse anti-α-tubulin antibodies (DM1A, Sigma), in combination with 1:200 rabbit anti-AIR-1 (Hannak et al., 2001 (link)). Embryos were fixed in methanol at −20°C for 30 min and incubated with primary antibodies for 1 h at room temperature. Secondary antibodies were Alexa488-coupled anti-mouse (Thermofisher, A11001) and Alexa568-coupled anti-rabbit (Thermofisher, A11011), both used at 1:500. Confocal images were acquired on a FV3000 confocal system with high-sensitivity cooled GaAsP detection unit (Olympus) using a 60× objective with NA 1.4 oil and processed in ImageJ and Adobe Photoshop, maintaining relative image intensities.

For endogenous protein expression analyses, cells were harvested with trypsin-EDTA, washed with cold phosphate buffered saline, and resuspended in RIPA buffer (10 mM Tris, pH 8.0, 140 mM NaCl, 0.1% sodium deoxycholate, and 1% (v/v) Triton X-100, 0.1% (w/v) SDS) supplemented with protease inhibitor cocktail (Pierce #A32965), for 1 h on ice. Clarified whole cell lysate was obtained by centrifugation at 12,000 × g for 15 min at 4°C. Mitochondrial isolation was performed using the ThermoScientific Mitochondrial Isolation Kit for Cultured Cells (#89874). Mitochondrial pellets were lysed with 2% CHAPS in Tris-buffered saline (TBS). Protein concentrations were determined by Bradford assay, and samples were stored at −20°C. Proteins (5μg) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore Immobilon®-PSQ membrane 0.2 μm). After blocking membranes with 5% milk solution, blots were probed with the following antibodies: anti-HSP60 (LK-2 clone, Enzo #ADI-SPA-807), anti-GAPDH (Poly6314 clone, Biolegend #631401), anti-COX-IV (3E11 clone, Cell Signaling #4850), Alex800 coupled anti-rabbit (ThermoFisher #A32735) and Alexa488 coupled anti-mouse (ThermoFisher #A11001). Proteins were visualized by fluorescence using a ChemiDoc MP Imaging system (Biorad) and densitometry analysis was performed using Image Lab software.