Ab208943

Paraffin-embedded tissue sections were deparaffinized, rehydrated, and antigen-retrieved, and cells were fixed and permeabilized. After blocking, they were incubated with antibodies against CitH3 (1:100, ab5103, Abcam), MPO (1:50, AF3667, R&D Systems, MN, USA), Ly6G (1:100, 127601, Biolegend, CA, USA), LC3B (1:100, ab192890, Abcam), LAMP1 (1:100, ab208943, Abcam), METTL3 (1:100, ab195352, Abcam), SIRT1 (1:100, ab110304, Abcam) and fluorescent secondary antibodies. Finally, the nuclei were stained with DAPI. The slices were viewed under an Olympus microscope (Tokyo, Japan).

N2a cells were transfected with indicated plasmids with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. At 48 h post transfection, the cells were washed three times with cold PBS and fixed with 4% (v/v) paraformaldehyde for 10 min at room temperature then washed three times with cold PBS. And the cells were permeabilized in PBS containing 0.5% Triton X-100 for 5 min at 4 °C, then blocked in 10% goat serum which were diluted with PBS for 2 h at 37 °C, and probed with primary antibodies which were diluted with PBS and 5% (w/v) BSA for 2 h at 37 °C. The primary antibodies were against Flag tag (MBL, M185-3L, 1:500) and LAMP-1 (Abcam, Ab208943, 1:100), then treated with Alexa Fluor 594-conjugated anti-rabbit antibody (Invitrogen, A11012, 1:500) or Alexa Fluor 488-conjugated goat anti-mouse antibody (Invitrogen, R37120, 1:500) as a secondary antibody for 1 h at 37 °C, and then stained with DAPI for 10 min. Cells were again washed three times with PBS, and then imaged with a ZEISS confocal microscope under oil objective.

After treatment, BV-2 cells were fixed with 4% paraformaldehyde for 20 min then permeated by 0.3% Triton X-100 for 10 min and subsequently blocked by 1% BSA for 20 min. Primary antibodies (galectin-3, ab76245, Abcam, 1:400; lysosomal associated membrane protein 1, LAMP1, ab208943, Abcam, 1:400; Lipid A, GTX40001, GeneTex, 1:400; cytosolic phospholipase A2-α, cPLA2, sc-454, Santa Cruz, 1:100; Cyt c, 66264–1, Thermo Fisher, 1:400) were incubated with cells overnight at 4°C. Fluorescence conjugated secondary antibodies (Jackson ImmunoResearch, 711-025-152, 705-095-147, 115-095-003, 715-585-150, 1:200) were added at room temperature for one hour.
After fixing with 4% paraformaldehyde, cells were stained with BODIPY™ 493/503 (Invitrogen, Life Technologies, Carlsbad, CA, USA) or PI (Beyotime, Shanghai, China) for 20 min at room temperature. TUNEL staining (MK1027, BOSTER, Wuhan, China) was performed following the manufacturer’s instructions.
Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI), and all images were captured with fluorescent microscopy (Olympus BX41).

N2a cells, the ipsilateral cortex of SD rats, and OM-MSCs were processed for western blotting as described previously. Proteins were transferred to the PVDF membrane (Millipore, USA) when the bromophenol blue reached the bottom, and the blots were blocked in 4% BSA in TBST (0.05%) solution for 1 h at room temperature and then incubated at 4°C overnight with the corresponding primary antibody. After incubation with secondary antibodies at room temperature for at least 1 h, the blot was visualized by the ChemiDoc XRS imaging system.
The primary antibodies were as follows: anti-caspase3 (19677-1-AP, Proteintech, USA), anti-GOLPH3 (ab98023, Abcam, Cambridge, UK), anti-SPCA1 (ab126171, Abcam, Cambridge, UK), anti-LC3 (ab192890, Abcam, Cambridge, UK), anti-LAMP1 (ab208943, Abcam, Cambridge, UK), anti-Akt (60203-2-Ig, Proteintech, Chicago, USA), anti-p-Akt (ab38449, Abcam, Cambridge, UK), anti-mTOR (20657-1-AP, Proteintech, Chicago, USA), anti-p-mTOR (ab109268, Abcam, Cambridge, UK), anti-PEDF (bs-20784R, Bioss, Beijing, China), and anti-β-actin (66009-1-Ig, Proteintech, Chicago, USA). The anti-rabbit and anti-mouse IgG secondary antibodies were obtained from Proteintech (Chicago, USA).

The paraffin-embedded liver tissue sections were cut into 10 μm coronal sections and used for immunofluorescence staining. These prepared sections were blocked with mouse serum and incubated with ADRP/perilipin2 (15294-1-AP, Proteintech, Wuhan) overnight at 4 °C, followed by fluorescence conjugated secondary antibody (Servicebio, Wuhan, China). The sections then added CY3-TSA (Servicebio, Wuhan) and were incubated in dark for 10min, followed by antigen retrieval with EDTA antigen retrieval buffer (1 mM EDTA, pH 8.0). Subsequently, these sections were incubated with the second primary antibody LAMP1 (ab208943, abcam), and followed by a fluorescence conjugated secondary antibody from Servicebio. The immunofluorescence was captured by using a Nikon Eclipse C1 microscope.

MC3T3-E1 cells were seeded in confocal dishes with different treatments, fixed with paraformaldehyde, and permeabilized with 0.2% Triton-X. After blocking with 5% BSA, cells were incubated with rabbit anti-TOM20 (CST, #42406) or rabbit anti-HSP60 (CST, #4870) overnight at 4 °C. After washing and incubating with FITC-labeled goat anti-rabbit IgG (Beyotime, China) or Cy3-labeled goat anti-rabbit IgG (Beyotime, China). For immunofluorescence co-localization, cells with different treatments were fixed, permeabilized and blocked as mentioned above, and incubated with rabbit anti-LAMP1 (abcam, ab208943) and mouse anti-COX IV (abcam, ab33985) overnight at 4 °C, and then incubated with Cy3-labeled goat anti-rabbit IgG (Beyotime, China) and FITC-labeled goat anti-mouse IgG (Beyotime, China). All of the cells were stained by DAPI (Abcam,Cambridge,UK), and observed by ZEISS LSM 880 (Carl Zeiss, Germany).

MDSCs (2 × 10⁶/well) were treated with G-SCF and GM-SCF for 48 h. Then, the selective lysosomal inhibitor NH4Cl (250 μM) or chloroquine (20 μM) was added, followed by incubation for 24 h at 37 °C with 5% CO₂. After incubation, the cells were washed three times with FACS buffer (0.5% FBS in 1 × PBS). The primary antibody, LAMP1 (ab208943, Abcam, 1:500) was added and incubated for 30 min on ice in the dark. Cells were washed three times by centrifugation at 1500 rpm for 5 min and resuspended in FACS buffer. Fluorochrome-labeled secondary antibodies Alexa Fluor®R555 (FcMACS, 1:1000) and streaming antibodies (CD11b, Gr1, and PD-L1) were added and incubated for 30 min on ice in the dark. Cells were then washed three times by pelleting at 1500 rpm for 5 min and resuspended in FACS buffer. Cells were analyzed using a FlowSight Imaging flow cytometer (Amnis). Data were analyzed using IDEAS software (Amnis).