Alexa fluor 488 and alexa fluor 546 conjugated secondary antibodies

Primary antibodies with the following specificities were used: HRD1/SYVN1 (C-term; Sigma-Aldrich, St. Louis, MO, USA), Sel1L (T-17; Santa Cruz Biotechnology), protein disulfide isomerase (PDI; RL90; Thermo Scientific Pierce Products), tau (Tau-5; Millipore, Bedford, MA, USA), γ-tubulin (GTU-88; Sigma-Aldrich), β-actin (C4; Santa Cruz Biotechnology), 4-hydroxy-2-nonenal (4-HNE; HNEJ-2; JaICA, Shizuoka, Japan). Conjugated secondary antibodies were anti-rabbit and anti-mouse immunoglobulin G (IgG)–horseradish peroxidase (HRP) (GE Healthcare, Buckinghamshire, UK), anti-goat IgG–HRP (Promega, Madison, WI, USA). Alexa Fluor 488- and Alexa Fluor 546-conjugated secondary antibodies were purchased from Molecular Probes (Eugene, Oregon, USA). Thapsigargin, tunicamycin, and hydrogen peroxide (H₂O₂) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Rotenone, HNE-DMA, and G418 were purchased from Sigma-Aldrich.

Cells were fixed with 4% (w/v) paraformaldehyde and washed. Subsequently, cells were permeabilized with 0.1% [v/v] Triton™ X-100 and blocked with PBS containing 1% (w/v) BSA and 5% (v/v) normal goat serum. After washing, cells were incubated with anti-ICAM-1 (BioLegend) and anti-SM22 (ab14106, Abcam) antibodies at 4 °C overnight. After washing, cells were stained with Alexa Fluor® 488- and Alexa Fluor® 546-conjugated secondary antibodies (Molecular Probes) and Alexa Fluor® 647-conjugated cholera toxin B subunit for GM1 detection, and then counterstained with DAPI. Immunofluorescence images were acquired using a confocal laser scanning microscope. For the quantification of SM22-positive cells, the numbers of SM22-positive cells with red color and the total numbers of cells stained with DAPI from four fields were counted and then the percentage of SM22-positive cells was calculated.

Cells were fixed with 4% (w/v) paraformaldehyde and then washed. Subsequently, cells were permeabilized with 0.1% (v/v) Triton™ X-100 and blocked with PBS containing 1% (w/v) BSA and 5% (v/v) normal goat serum. Following washing, the cells were incubated with anti-GD1a and monoclonal mouse anti-α-SMA (ab7817; Abcam) anti-bodies at 4°C overnight. The cells were washed, stained with Alexa Fluor^® 488- and Alexa Fluor^® 546-conjugated secondary antibodies (Molecular Probes), and counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI). Immunofluorescence images were acquired using a confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany).