Dab quanto kit

Macrophage contents in atherosclerotic lesions were measured using immunohistochemistry staining. Briefly, frozen aortic sinus sections were incubated with 3% H₂O₂ for 10 min, blocked with 3% BSA (Siama) for 1 h, and incubated with anti-F4/80 antibody (1:200, Abcam, Inc., CA, USA; Cat.No. ab-300421) overnight at 4 °C. After incubation with anti-rabbit lgG for 1 h at room temperature, the slides were developed with 3,3-diaminobenzidine (DAB Quanto Kit, TA-060-QHDX, ThermoFisher) and stained with hematoxylin. Images were recorded uding a light microscope.

For immunofluoresence staining, cells were seeded in confocal dish for 24 h with or without CQ treatment and CsA pretreatment in some cases. The cells were fixed with 4% paraformaldehyde in PBS pH 7.4 for 10 min at room temperature and then permeabilizated with 0.5% Triton X-100 in PBS for 10 min. After blocking with 2% BSA in PBS containing 0.1% Tween-20 for 30 min, cells were incubated with NF-κBp65 (Cell Signaling Technology, #8242, 1 : 400), TFEB (Bethyl Laboratories, #A303-673A, 1 : 500), Lamp1 (Abcam, ab25245, 1 : 500), and Lamp2 (Abcam, ab25339, 1 : 100) in 2% BSA in PBS containing 0.1% Tween-20 overnight at 4 °C. After washing and staining with secondary antibody for 1 h at room temperature, nucleus were stained with DAPI (4',6-diamidino-2-phenylindole) (2 μg ml⁻¹). The merged figures were analyzed by OLYMPUS two-photon microscope. For immunohistochemical staining, melanoma tissues were isolated from tumor-bearing mice and fixed in 37% formalin and embedded in paraffin. Next, the sections was incubated with the anti-Granzyme B (Abcam, ab4059, 1 : 1,000) by using the UltraVision Quanto Detection System kit (Thermo Scientific) and observed by using the DAB Quanto kit (Thermo Scientific).