Anti lc3b d11 xp rabbit mab

IF microscopy staining for localization and quantification of autophagy proteins LC3B, SQSTM1/p62, and ATG5 in situ was performed with anti-LC3B (D11) XP Rabbit mAb (Cell Signaling Technology), anti-SQSTM1/p62 (abcam), and anti-APG5L/ATG5 (EPR1755) (abcam) antibodies. Briefly, 5-μm-thick cryosections were fixed with cold acetone (−20 °C) for 10 min. After several washes in PBS, primary antibodies were incubated overnight at 4 °C. Alexa Fluor 555-conjugated donkey anti-Rabbit and Alexa Fluor 488-conjugated donkey anti-Mouse (Thermo Fisher) were adopted as secondary antibodies. Confocal images of HF derma papilla and of the area surrounding were taken by using a Confocal Microscope NIKON A1, the 20 × 0.25 and 60 × 1.40 numerical aperture objective lens. Quantitative immunohistomorphometry in defined reference area using standardized light exposure was performed with Image J (NIH) software, as described [29 (link),64 (link)]. Microscopic hair cycle staging was performed as previously described, using Ki-67/TUNEL immunostaining and Masson Fontana histochemistry [15 (link),17 (link),29 (link)].

Cultured MDA-MB-231 and MCF-10F cells were gently scraped, pelleted by centrifugation at 5000× g for 5 min at 4 °C, resuspended and lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors. For Western blot, 60 μg of protein were subjected to 10% SDS-polyacrylamide electrophoresis. Proteins were then transferred to nitrocellulose membranes (Thermo Fisher) and incubated with primary antibodies against TASK-3 (1:1000 antibody dilutions).
Membranes were then incubated with 1:5000 peroxidase-conjugated secondary antibodies and antibody-antigen complexes were then visualized using ECL Plus Kit and a hyper film MP (GE Healthcare, Little Chalfont, UK). Quantification of protein levels in the Western blots was performed using ImageJ software version 1.48d (National Institutes of Health, Bethesda, MD, USA). The following antibodies were used in this study: goat anti-TASK-3 (sc-11317; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LC3B (D11 XP rabbit mAb; Cell Signaling Technology, USA), anti-cleaved caspase-3 (Asp175 5A1E rabbit mAb; Cell Signaling Technology, Danvers, MA, USA), anti-Rb (D20 rabbit mAb; Cell Signaling), anti-Phospho-Rb (D59B7 rabbit mAb; Cell Signaling), mouse anti-GAPDH (sc-365062; Santa Cruz) and a secondary rabbit anti-goat HRP (sc-2768; Santa Cruz).