Anti lamp2 antibody

HT29 and HCT116 cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% TrintonX-100 in DPBS, and blocked with 3% goat serum. Then cells were stained with anti-GFP, anti-LC3B (Cell Signaling Technology), and anti-LAMP2 antibody (Santa Cruz Biotechnology), and counterstained with DAPI (Thermo Fisher Scientific, USA). Image taking and processing were carried out with laser scanning confocal microscopy (Carl Zeiss AG, Germany). Visualization and picture in the same panel were taken under the same excitation conditions. Fresh frozen tumor tissues were sectioned at 10 μm with a cryostat. Anti-CD8, anti-PD-L1, anti-CD8, and DAPI were used for detection.

Cells, after growing overnight on coverslips to required confluence, were treated with TN‐16 for indicated time periods and fixed with 4% paraformaldehyde. Cells were subsequently permeabilized (0.5% Triton X100 in PBS), blocked with 2% BSA in PBS and incubated overnight with primary antibodies against specific target proteins at 4°C. Following primary antibodies were used at indicated dilutions for probing target proteins—anti‐LC3A/B antibody (Cell Signaling Technology, 9091; 1:250), anti‐LAMP‐2 antibody (Santa Cruz Biotechnology, sc‐18822; 1:250) and anti‐β‐tubulin (Thermo Fisher Scientific, 480011; 1:250). After 3 × 5 minutes washes in PBS, cells were incubated with corresponding (anti‐mouse Alexa Fluor™ 488, A10468; anti‐mouse Alexa Fluor™ 594, A11032 or anti‐rabbit Alexa Fluor™ 594, A11037) fluorescence‐conjugated secondary antibody (Invitrogen Corp) @1:250 dilutions for 1 hour at room temperature (RT). Following three washes, coverslips were mounted on glass slides using ProLong Gold Antifade reagent (Invitrogen Corp) containing DAPI. Samples were subsequently examined using appropriate excitation and emission filters under LSM510 META confocal microscope (Carl Zeiss) and a Plan Apochromat 63 × 1.4 NA Oil DIC objective lens.

Animals were perfused, and immunohistochemical procedures were performed as described later. Rapidly frozen sections with 20 μM were co-incubated with primary anti-AIF1 antibody (Wako Pure Chemical Industries, Chuo-ku, Osaka, Japan, 019-19741) and anti-LAMP2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA, sc-19991) overnight at 4°C. Secondary AlexaFluor 488 goat anti-rabbit IgG (A-11008) or AlexaFluor 594 goat anti-mouse (A-11032) from Thermo Fisher Scientific Waltham, MA, USA, was added for 2 h to detect Iba1 and LAMP2, followed by mounting of sections with prolong gold antifade reagent with 4,6-diamidino-2-phenylindole (Thermo Fisher Scientific, Waltham, MA, USA, P36935). Fluorescent images were acquired on a Zeiss Observer. AxioVs 40 4.8.0.0 software (Carl Zeiss, Thornwood, NY, USA) was used to process the images.

Antibodies against phosphorylated PKCδ (Thr507), AMPKα 1/2 (Thr172) and anti- LAMP-2 antibody were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The anti-phosphorylated mammalian target of rapamycin (mTOR) (Ser2448) antibody was obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Antibodies against NeuN, CatB and CysC were obtained from EMD Millipore Corp. (Billerica, MA, USA). Alexa Fluor-conjugated secondary antibodies were purchased from Life Technologies Corp. (Grand Island, NY, USA). We also used the following commercially available antibodies: anti-c-myc (Roche, Basel, Switzerland), anti-tubulin, anti-β-actin (both from Sigma-Aldrich Co. LLC St. Louis, MO, USA) and anti-LC3 (Novus Biologicals LLC, Littleton, CO, USA). Rabbit anti-human SOD1 was raised in our laboratory against a recombinant human SOD1 peptide (24–36) and purified with protein A.^{51 (link)}

The following primary antibodies were used in our experiments: anti‐GAPDH antibody (Proteintech), anti‐Histone H3 antibody (Proteintech), anti‐phospho‐p70S6K antibody (T389) (Cell Signaling Technology), anti‐p70S6K antibody (Epitomics), anti‐LAMP1 antibody (Abcam), anti‐LAMP2 antibody (Santa Cruz), anti‐TFEB antibody (Cell Signaling Technology), anti‐p62 antibody (Enzo Life Sciences), anti‐mTOR antibody (Cell Signaling Technology), anti‐HA antibody (Santa Cruz), anti‐FLAG antibody (Sigma), anti‐GFP antibody (Santa Cruz), anti‐Rag B antibody (Cell Signaling Technology), anti‐raptor antibody (Cell Signaling Technology), anti‐α‐Tubulin (Proteintech), and anti‐C9orf72 (Santa Cruz). The following secondary antibodies were used: horseradish peroxidase‐conjugated sheep anti‐mouse and anti‐rabbit antibodies (Jackson ImmunoResearch Laboratories). The following fluorescent secondary antibodies were used: Alexa Fluor 594‐conjugated goat anti‐rabbit IgG (Proteintech), Alexa Fluor 594‐conjugated goat anti‐mouse IgG (Proteintech), Alexa Fluor 488‐conjugated goat anti‐rabbit IgG (Proteintech), Alexa Fluor 488‐conjugated Goat anti‐mouse IgG (Proteintech), Alexa Fluor 660‐conjugated goat anti‐mouse IgG (H + L) highly cross‐adsorbed antibody (Invitrogen), and Alexa Fluor 405‐conjugated goat anti‐rabbit IgG (H + L) secondary antibody (Invitrogen).