Anti lamp 1 cd107a antibody

Immunofluorescence staining was performed on fixed RANKL‐primed BMDMs, blocked with 5% BSA and incubated overnight with anti‐Galectin‑3 mAb (R&D Systems, Cat# AF1197) and anti‐LAMP‐1 (CD107a) Antibody (Sigma Aldrich, Cat# AB2971). The cells were, then, incubated with Alexa Fluor^® 488 Goat anti‐Rat IgM Antibody (Biolegend, Cat# 408908), Alexa Fluor^® 488 Donkey Anti‐Goat IgG H&L (Abcam, Cat# ab150129) and Alexa Fluor^® 405 Goat Anti‐Rabbit IgG H&L (Abcam, Cat# ab175652). The nuclei were counterstained using Draq5^TM (Thermo Scientific, Cat#62254) staining solution, and the slides were mounted using Aqua‐Poly/Mount (Polysciences, Cat# 1860620). The images were acquired using a Zeiss LSM 800 confocal microscope at 20 and 40× magnification and analyzed using the Zen (Black edition), Zen 3.2 (Blue edition) and Image J software.

RANKL‐stimulated RAW264.7 cells were lysed in CytoBuster^TM Protein Extraction Reagent (Millipore, Cat# 71009) supplemented with protease and phosphatase inhibitors. The lysates were denatured, separated in a Bolt™ 4–12% Bis‐Tris gel (Invitrogen, Cat# NW04120BOX) and transferred to a membrane using an iBlot™ 2 PVDF Gel Transfer Stack (Invitrogen, Cat# IB24001). The membrane was blocked with 5% Blotting Grade Blocker Non Fat Dry Milk (BioRad, Cat# 1706404XTU) and incubated overnight with Human/Mouse/Rat Galectin‑3 Antibody (R&D Systems, Cat# AF1197), Anti‐LAMP‐1 (CD107a) Antibody (Sigma Aldrich, Cat# AB2971) and GAPDH (14C10) Rabbit mAb (Cell Signaling Technology, Cat# 2118) in blocking buffer. The cells were, then, incubated with HRP‐linked secondary antibodies, and the protein bands were detected using the chemiluminescent HRP substrate SuperSignal™ West Pico PLUS (Thermo Scientific, Cat# 34579).