Fv500 confocal laser scanning microscope

For immunofluorescence staining, the frozen sections were permeabilized with 0.3% Triton X-100 and blocked with 5% (vol/vol) bovine serum albumin (BSA) in PBS. Then the sections were incubated with primary antibodies, including anti-S100B antibody (1:500, AMAb91038, Atlas Antibodies AB, Stockholm, Sweden), anti-ALDH3B2 antibody (1:50, HPA045132, Atlas Antibodies AB, Stockholm, Sweden), Anti-8-Oxoguanine (8-OxoG) Antibody (1:100, MAB3560, Millipore, Sigma-Aldrich Co. LLC., CA, United States) at 4°C overnight. Secondary antibodies were either FITC- -conjugated goat anti-mouse IgG antibody (1:100, 115-095-003, Jackson ImmunoResearch Laboratories, Inc., PA, United States), Alexa Fluor^® 594-conjugated goat anti-rabbit IgG antibody (1:50, 111-585-003, Jackson ImmunoResearch Laboratories, Inc., PA, United States) or Alexa Fluor^® 594-conjugated goat anti-mouse IgG antibody (1:50, 115-585-003, Jackson ImmunoResearch Laboratories, Inc., PA, United States), and incubated for 2 h in dark at RT. After washing in PBS, the sections were mounted with DAPI-containing mounting media (C1211, APPLYGEN, Beijing, China). Immunofluorescence reactivity or DAPI was detected by Olympus IX71 microscope and Olympus FV500 confocal laser-scanning microscope (Olympus, Japan), and analyzed with Image J 1.46r.

The experimental procedure was based on previously published protocols (Cisbani et al., 2013 (link); Cicchetti et al., 2014 (link)). In brief, prior to immunostaining, sections were deparaffinized in Citrisolv™ (Fisherbrand), rehydrated and incubated overnight at room temperature in blocking solutions containing the primary antibodies listed in Supplementary Table 1. After KPBS washes, sections were incubated with appropriate secondary antibodies in a blocking solution for 2 h at room temperature (Goat anti-mouse Alexa Fluor® 488, 1:750; Goat anti-rabbit Alexa Fluor® 546, 1:500; Goat anti-chicken Alexa Fluor® 647, 1:500; all Life Technologies).
Following DAPI incubation (2 mg/ml, Molecular Probes), sections were washed and coverslipped with Fluoromount-G™ (SouthernBiotech). Confocal laser scanning microscopy was performed using an Olympus FV500 confocal laser-scanning microscope. Images were acquired by sequential scanning and optimized by a two-frame Kalman filter and analysed using acquisition software from Olympus (Fluoview SV500 imaging software 4.3, Olympus) and ImageJ (NIH).

The experiments were done as previously described.21 Briefly, sterile coverslips coated with poly‐l‐lysine (BD Biosciences) were washed with PBS and incubated with 0.5% glutaraldehyde for 15 minutes at room temperature. Coverslips were washed with PBS and coated with FITC‐conjugated gelatin (Invitrogen) for 10 minutes. After washing with PBS, coverslips were incubated with sodium borohydride for 1 minute and washed with PBS. Cells were seeded in 24‐well plates containing prepared coverslips coated with fluorescent gelatin and incubated at 37°C for 12 hours. Next, cells were fixed with 4% formaldehyde and labelled for filamentous actin with Alexa Fluor 568‐phalloidin. Images were taken using the Olympus FV500 confocal laser scanning microscope and FluoView software (Olympus). Sites of degraded matrix were visible as dark areas (spots) in the bright green fluorescent gelatin matrix. The area of gelatin digestion was calculated for 40 cells per condition using ImageJ software.20