Image pro express program

Fibroblasts and primary neurons were seeded on poly-lysine-coated coverslips (30,000 cells/cover). After treatment, cells were fixed in 4% paraformaldehyde/4% sucrose in PBS and permeabilized with 0.02% Triton X-100. Then, cells were blocked with 3% bovine serum albumin in PBS. Immunostaining was carried out using anti-tyrosine 412 phosphorylated of c-Abl (Y412) (anti-p-c-Abl) (C5240, Sigma Chemical co), anti-p62 (ab56416; Abcam), anti-LAMP1 (1D4B, sc-19992; Santa Cruz Biotechnology). Anti-rabbit IgG conjugated with Alexa Fluor-488 and anti-mouse IgG conjugated to Alexa Fluor-555 and Hoechst 33342 (H3570) were obtained from Invitrogen Detection Technologies. Fluorescent images were captured with an Olympus BX51 microscope (Olympus, Tokyo, Japan) and analyzed with the Image-Pro Express program (Media Cybernetics). We examined at least five images by cover and three covers by condition were stained by experiment in at least three independent experiments.

Two-micron sections of gallbladder samples were fixed with formalin and embedded in paraffin. For TRAF3 immunohistochemistry analysis, the rabbit anti-TRAF3-ab155298 antibody was used (1:100, ABCAM, Ltd, Cambridge, UK) and was visualized using the Envision Flex/HRP SM802 complex method (Dako, Agilent Technologies Company, Santa Clara, CA 95051, USA). Labeled sections were examined and captured using an Olympus BX51 microscope (Olympus, Tokyo, Japan) with the software Stereo Investigator v.11.03 (MBF bioscience, Williston, VT, USA) and analyzed with the Image-Pro Express program (Media Cybernetics, Bethesda, MD, USA) as described previously^{66 (link)}.