4.6. Immunocytochemistry (ICC)

Sequential immunocytochemistry was performed to evaluate the effect of Cd exposure on protein expression and cellular localization of specific proteins. WT and HD cells were seeded at 40,000 and 50,000 cells, respectively, and incubated overnight at 33 °C in 8-well-chambered slides. Following exposure to 40 µM CdCl₂ for the indicated time points, cells were fixed with 4% PFA for 30 min, washed with 1X PBS for 5 min three times, permeabilized with 0.01% Triton X-100 for 10 min, washed three times with 1X PBS for 5 min each, and blocked with 3% BSA for 30 min. Following blocking, cells were incubated with the first primary antibody in 3% BSA overnight at 4 °C and shaken at 2.5 rpm. Primary antibodies (ABclonal rabbit monoclonal anti-dynamin related protein (DRP1, A2586), mitochondrial fission protein 1 (FIS1, A19666), mitofusin 1 (MFN1, A9880), mitofusin 2 (MFN2, A12771), light chain 3-I/II (LC3-I/II, A5618), p62 (A19700), autophagy-related 5 (ATG5, A0203), and cytochrome C (Cyto. C, A13430)) were used at 1:100. The cells were then washed with 1X PBS for 5 min three times and incubated with Alexa Fluor 594- or 488-conjugated secondary antibodies for 1 h. For the second round of staining, cells were washed with 1X PBS for 5 min three times and re-blocked in 3% BSA for 30 min and subjected to the above staining procedure for the second round of antibodies. The cells were mounted in an antifade mounting medium (Aqua-Poly/Mount, Polysciences, Warrington, PA, USA, 18606-20) containing DAPI (VWR, IC15757410; 1:1000). Fluorescent images were acquired using a Leica DM4000B fluorescent microscope or a Zeiss LSM 880 confocal microscope at 63× magnification. Each fluorescent channel was captured in a Z-series of 6 slices to eliminate the detection of bleed-through and other artifactual fluorescence.