Sa00013 1

Cells were cultured on poly‐lysine coated dish overnight. Immunofluorescence staining was then performed according to a previous study.^[47] In brief, permeabilized cells were processed in blocking solution (0.1% Triton X‐100 and 5% BSA in PBS) for 1 h at room temperature. Then, the samples were treated with 1.5 m HCl for 1 h. After washing with PBS, cells were incubated with 5‐methylcytosine primary antibodies (1:500, proteintech, 61480) at 4 °C overnight. Subsequently, the samples were incubated with the fluorescence tagged secondary antibody (1:500, proteintech, SA00013‐1) for 1 h at room temperature. Following staining with Hoechst 33342, images were captured by Olympus microscopy.

The mouse brain sections (6 μm) were incubated with primary antibodies against NOX2 (1:200, ab133303, Abcam), DHFR (1:200, ab124814, Abcam), GFAP (1:200, 2389127, Invitrogen, Carlsbad, CA, USA), and CD31 (1:200, ab24590, Abcam) at 4°C overnight. GFAP is considered a standard marker of mature astrocytes and it is often used to label astrocytes in IF. CD31 is also commonly used to label endothelial cells in IF. So, we used CD31 as a marker to distinguish endothelial cells and GFAP as a marker to distinguish astrocytes. The brain slices were incubated using the corresponding goat anti-rabbit IgG antibodies (1:250, SA00009-2, Proteintech) or goat anti-mouse IgG (1:250, SA00013-1, Proteintech) for 1.5 h at room temperature. Subsequently, the brain sections were sealed with anti-fluorescence solution after incubated DAPI (C1005, Beyotime) for 6 min. Finally, the sections were observed and captured under the fluorescence microscope. Immunofluorescence staining of hCMEC/D3 and HA was similar to brain sections after fixing cells with 4% paraformaldehyde.

The cells were fixed in 4% Paraformaldehyde, permeated with 0.1% Triton X-100, and then incubated at 4 °C overnight with primary antibodies against O-GlcNAc and COX10. Then, the cells were incubated with CL594 coupled anti-rabbit (1:500, SA00013-4, Proteintech) or CL488 coupled anti-mouse IgG (1:500, SA00013-1, Proteintech) secondary antibodies, and the nuclei were stained with DAPI. Finally, the immunofluorescence-stained cells were imaged using an Olympus BX51 immunofluorescence microscope.

The mice were anesthetized 1 hour after the last METH/saline injection. The brains were post-fixed in the perfusion solution for 4 h at 4 °C followed by 24 hours of incubation in 30% sucrose solution. The Tissue-Tek O.C.T. compound (Sakura, Japan) was used to embed brain tissue, which was consequently cut in 20-μm slices using a cryostat (Leica, Germany). Brain sections were blocked with 5% bovine serum albumin containing 0.3% Triton X-100 at 37 °C for 60 min and incubated with primary antibodies to rabbit anti-Notch1 (1:200, Proteintech, 20687-1-AP); mixed rabbit anti-Notch1 and mouse anti-GABA_B1 (1:100, Abcam, ab55051) overnight at 4 °C. Subsequently, brain tissue sections for Notch1 receptor were incubated with goat anti-rabbit coralite 488 conjugate (1:500, Proteintech, SA00013-2); brain tissue sections for Notch1 and GABA_B1 receptor double staining were incubated with goat anti-rabbit coralite 594 conjugate (1:500; SA00013-4; Proteintech) and goat anti-mouse coralite 488 conjugate (1:500; SA00013-1; Proteintech) for 1 h at room temperature. Nuclei in immunolabeled specimens were visualized with DAPI. The sections were observed under a fluorescence microscope (Carl Zeiss, Axio scope A1, Germany). At least 10 regions from three mice of each group were chosen randomly and analysed by observers who were unaware of the experimental cohort.

Brain tissues were fixed, dehydrated, and then cut into coronal sections at a thickness of 10 μm. Sections were permeabilized with 0.3% Triton X-100. Then, brain slices were blocked with 5% donkey or goat serum for 1 h and incubated overnight at 4°C with primary antibodies: ABIN1 (bs-9568R, Bioss, China, 1 : 50), NeuN (MAB377, Millipore, Germany, 1 : 200), A20 (3A11G6, Proteintech, USA, 1 : 100), Iba-1 (NB100-1028, Novus, USA, 1 : 50), and GFAP (BM0055, Boster, China, 1 : 100). Then, the following fluorescently labeled secondary antibodies were incubated with the sections at 37°C for 1 h in the dark: CoraLite594-goat anti-rabbit (SA00013-4, Proteintech, 1 : 200), CoraLite488-goat anti-mouse (SA00013-1, Proteintech, 1 : 200), CoraLite594-donkey anti-rabbit (SA00013-8, Proteintech, 1 : 200), FITC-donkey anti-goat (SA00003-3, Proteintech, 1 : 200), and Cell nuclei were stained with DAPI. Brain slices were observed under a laser confocal microscope (LSM-800, Carl Zeiss Micro-Imaging Co., Germany).

HUVECs were fixed with 4% paraformaldehyde for 30 min, washed with PBS for 5 min, and permeabilized in 0.1% Triton X-100 at room temperature for 10 min. The cells were incubated with rabbit polyclonal antibody against HO-1 (1:100), mouse monoclonal antibody against NPM1 (1:100), rabbit polyclonal antibody against fibrillarin (1:100) or mouse monoclonal antibody against p53 (1:50, Cell Signaling Technology, #2524) at 4 °C overnight. The cells were then washed with PBS and incubated with appropriate secondary antibodies in goat serum (Boster Biological Technology, California, USA) for 1 h at room temperature. Alexa Fluor 594-conjugated anti-rabbit IgG (H+L) secondary antibody and Alexa Fluor 488-conjugated anti-mouse IgG (H+L) secondary antibody (Proteintech, SA00013-4, SA00013-1) were used. Nuclei were stained with 5 μg/ml DAPI (Sigma). Images were acquired using cell auto imaging system (EVOS FL Auto, Life Technologies) or FV3000 laser scanning confocal microscope (Olympus Life Science).

The sections of pancreatic tissues were blocked by 10% goat serum (Servicebio, Wuhan, China) and incubated with primary antibodies at 4°C overnight. The primary antibodies used in this experiment were: rabbit anti-insulin (1:200; bs-0056R; Bioss), mouse anti-insulin (1:200; BM0080; Boster), rabbit anti-podoplanin (1:200; A01124-2; Boster), mouse anti-Fos (1:100; sc-166940; Santa Cruz), mouse anti-Bad (1:100; sc-8044; Santa Cruz). The secondary antibodies (SA00013-1, SA00013-2, SA00013-3, SA00013-4) for immunostaining were all purchased from Proteintech. 4′,6′-diamidino-2-phenylindole (DAPI; Servicebio) was used for nuclei staining. Olympus FV1000 confocal microscopy system was used for images capture.