Mouse anti tom20

For immunofluorescence microscopy, cells were grown on coverslips coated with PLO/laminin. Cells were washed with phosphate-buffered saline (PBS) with calcium and magnesium (PBS+/+) and fixed with 4% paraformaldehyde (PFA) for 10 min on ice. Subsequently, cells were washed with PBS+/+ and incubated with permeabilization buffer containing 0.1% Triton X-100 in PBS for 10 min on ice. After a washing step with PBS+/+, cells were incubated with 1% normal goat serum (NGS, Santa Clara, CA, USA) for 1 h at room temperature, followed by incubation with primary antibodies in 3% NGS at 4 °C overnight. The antibody used was mouse anti-Tom20 (1:100, Santa Cruz Biotechnology). Next, cells were washed with PBS+/+, followed by incubation with antimouse IgG conjugated with the Alexa488 secondary antibody (Invitrogen, Carlsbad, CA, USA) diluted 1:100 for 2 h at room temperature. After washing with PBS+/+, nuclei were stained with DAPI (5 min, 250 ng/mL). Cover slips were mounted using Fluoromount-G^® (SouthernBiotech, Birmingham, AL, USA). Microscopy was performed using an LSM 900 laser scanning microscope (LSM) equipped with a 130 × 100 STEP motorized scanning stage; a URGB laser module with a 405 nm, 488 nm, and 561 nm diode laser; a Plan-APOCHROMAT 63×/1.4 oil objective, and a GaAsP-PMT detector, using the ZEN 2 blue edition imaging software (all Zeiss, Hamburg, Germany).

Cells plated on coverslips in 12-well plates and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% v/v Triton X-100/1xPBS (10 min), and blocked with 10% normal goat serum prior to incubation with primary antibodies overnight. Rabbit anti-ki67 (Millipore, 1:200), rabbit anti-Tom20 (Thermofisher, 1:1000), mouse anti-cytochrome C (BD, 1:500), rabbit anti-APP (Cell sig, 1:200), mouse anti-P62 (abcam, 1:500) and mouse anti-Tom20 (Santa Cruz, 1:100). Cells were then labeled with appropriate secondary antibody raised in goat (Invitrogen). Images were taken with the LSM800 (Zeiss) confocal microscope by using the 40×/0.5 EC Plan-Neofluar objective or the 63×/1.4 Oil Plan-Apochromat objective. Excitation and acquisition parameters were constrained across all paired comparisons. For fluorescent quantification, morphometric measurements of images were performed using Image-Pro Plus software (MediaCybernetics). Mitochondrial morphology quantification was conducted by the Mito-Morphology Macro^{44 (link)} through ImageJ software (National Institutes of Health) as previous described^{43 (link)}. In brief, images of 30 cells from random view field of each group were first processed with a median filter to obtain isolated and equalized fluorescence, then individual mitochondria were analyzed for the lengths of major axes.

Primary antibodies used in this study: rabbit anti-LC3A/B, rabbit anti-Parkin rabbit anti-HSP60 (1:1000, Cell Signaling, products #4108S, #4211S and #12165S , respectively); mouse anti-TIM23 (1:1000, BD Biosciences, product #611222); mouse anti-VDAC1, mouse anti-TOM20, and mouse anti-p62 (Santa Cruz, 1: 500, sc-58649, sc-17764, and sc-28359, respectively); rabbit anti-ATP synthase gamma (1:1000, GeneTex, product #GTX-114275), mouse anti-Actin (1:3000, Genescript, product #A00702) and rabbit anti-GFP (Genescript, product #A01388-40). Secondary antibodies were: HPR conjugated goat anti-rabbit and goat anti-mouse (1:2500, products #170-6515 and #172-1011, respectively). CCCP and DMSO were obtained from Sigma. Bafilomycin A1 was obtained from LC Laboratories. Mitotracker Deep Red FM, Lysotracker Red DND 99, and TMRE, and Hoechst 33342 were obtained from Life Technologies. 3-Hydroxy-1,2-dimethyl-4(1H)-pyridone was from Sigma.