Bl105a

Ki67 immunofluorescence staining of cells cultured on coverslips was done to measure BMMSCs proliferation post-OGD. Cells were gently washed twice with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, and then rewashed thrice with PBS (5 min each). They were then permeabilized with 0.5% Triton X-100 for 20 min, washed thrice with PBS (5 min each), and blocked with 5% bovine serum albumin (BSA; Biofroxx GmbH, Hessen, Germany) for a minimum of 30 min. The blocking solution was discarded, and cells incubated with anti-Ki67 primary antibody (rabbit polyclonal, Proteintech, 27309-1-AP, 1:200) overnight at 4° C, and then with goat anti-rabbit IgG (H+L) DyLight 488 secondary antibody (Bioworld, BS10017, 1:500). The nuclei were stained with DAPI solution (Biosharp, BL105A) and imaged with an inverted fluorescence microscope (Leica Microsystems, Germany). The rate of proliferation was evaluated as the ratio of Ki67-positive (+) cells to the total number of cells (DAPI (+)) per field using the ImageJ software (NIH). For each slide, five random views were captured and averaged for statistical analysis.

Five hundred thousand B16-F10 cells were seeded in the circle microscope cover glasses and cultured overnight. CBD (Ca²⁺ (high)) at a CBD concentration of 0, 2.5, and 4 µg/mL were co-incubated with the cells for 2 h. After the removal of the medium, the cover glasses were fixed with 4% paraformaldehyde. Then, the cells were then permeabilized with Triton X-100 (0.1%, v%) for 15 min, followed by the addition of a blocking solution consisting of bovine serum albumin (BSA, 1%) and incubated at room temperature for 30 min. Next, all samples were incubated with the primary antibodies at room temperature for 12 h, followed by staining with the corresponding secondary antibodies in dark for 1 h. The primary antibodies included ATF3(ab207434, Abcam) and NFATc1(sc-7294, SANTA CRUZ). The following secondary antibodies included IgG (H + L) Fluor 555-conjugated (A0460, Beyotime) and IgG (H + L) Fluor 647-conjugated (A0468, Beyotime). Nuclei were labeled with DAPI (BL105A, Biosharp) at room temperature for 5 min. After washing with PBS thoroughly, immunofluorescence images were then acquired with a multiphoton confocal microscopy (STELLARIS 8 DIVE, Leica).

Cells were grown on glass coverslips (CITOTEST, 12-545-100) followed by fixation using 4% paraformaldehyde (Biosharp, BL539A) for 15 min at room temperature. The fixed cells were rinsed three times with PBS, permeabilized with 0.5% Triton X-100 for 30 min (for HEK293T cells, permeabilized for 10 min) and blocked with 10% FBS in PBS for 1 h at room temperature (RT), followed by incubation with primary antibody (anti-p62 antibody, dilution 1:500 (MBL, PM045); anti-LC3A/B antibody, dilution 1:100 (CST, 12741S); anti-Ub antibody (FK2), dilution 1:100 (MBL, D058-3); anti-HA antibody, dilution 1:100 (Santa Cruz, sc-7392)) diluted in PBS overnight at 4°C. Then, the cells were rinsed three times with PBS containing 0.01% Tween 20 and incubated with secondary antibody (Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L), Alexa Fluor 647-labeled goat anti-rabbit IgG (H+L), Alexa Fluor 488-labeled goat anti-mouse IgG (H+L) or Alexa Fluor 647-labeled goat anti-mouse IgG (H+L), dilution 1:500). For nucleus staining, cells were incubated with DAPl (Biosharp, BL105A) in room temperature for 5 min, and were then rinsed three times with PBS containing 0.01% Tween 20.

IHC staining was performed on paraffin‐embedded sections (5 µm) of the AAA/TAA/AD tissues as previously described.¹¹ Briefly, sections were incubated overnight with primary antibodies targeting ATP1A1 and ATP1A2 (1:1000, rabbit, 14418–1‐AP/16836‐1‐AP, Proteintech), followed by enhancer solution and biotinylated anti‐IgG and streptavidin‐peroxidase for 30 min at 37°C. Development of the chromogenic colour reaction was accomplished using the peroxidase substrate 3,30‐diamminobenzidine (Maixin, Fuzhou, China) after sections were washed with phosphate‐buffered saline (PBS) three times. IF staining was performed on paraffin‐embedded sections (5 μm) of AAA tissues. Sections were incubated overnight with primary antibodies against ATP1A1/ATP1A2 (1:100, rabbit, 14418–1‐AP/16836‐1‐AP, Proteintech) and α‐SMA (1:200, mouse, ab7817, Abcam), followed by donkey anti‐rabbit Alexa Fluor 568 (A10042, Invitrogen) and donkey anti‐mouse Alexa Fluor 488 (A21202, Invitrogen) secondary antibodies after the sections were washed three times with PBS. Nuclei were counterstained with 4′, 6‐diamidino‐2‐phenylindole (DAPI, BL105A, Biosharp). Images were captured using an Olympus BX51 TRF Fluorescent/light microscope system (Olympus, Tokyo, Japan).

Marc-145 cells treated with different conditions were collected and immediately treated with 4% paraformaldehyde (BL539A, Biosharp) for 20 min. Cells were permeabilized in 0.1% Triton X-100 (BS084, Biosharp) for 15 min and then incubated with 5% bovine serum albumin for 1 h. After blocking nonspecific binding, cells were incubated with specific antibodies overnight at 4°C. The cells were washed and incubated with secondary antibodies for 1 h. The LDs were stained with BODIPY493/503 for 10 min, the nuclei were stained with DAPI (BL105A, Biosharp) for 10 min. Finally, all samples were analyzed using the Leica TCS SP8 fluorescence microscope (Leica, Germany).

The cells were fixed with 4% formaldehyde (BL539A, Biosharp, Beijing, China) for 20 min and then incubated with 0.1% Triton X-100 (P0097, Beyotime Biotechnology, Shanghai, China) at room temperature for 20 min. After being blocked with 5% BSA for 1 h at room temperature, the cells were incubated with human anti-HIF-1α antibody (1:500, ab51608, Abcam, Cambridge, UK) at 4 °C overnight. After being washed 3 times with PBS, the cells were incubated with Goat Anti-Rabbit IgG(H + L) Fluor 594-conjugated (1:200, S0006, Affinity Biosciences, Cincinnati, OH, USA) in the dark for 1 h at room temperature. Finally, the nucleus was stained with stained with DAPI (BL105A, Biosharp, Beijing, China) for 5 min at room temperature. After being washed 3 times with PBS, the cells were observed under a fluorescence microscope (AF6000, Leica, Wetzlar, Germany).