C0060

Mice were deeply anesthetized and perfused transcardially with 0.9% normal saline followed by 4% PFA in PBS. Brains were postfixed in 4% PFA in PBS at room temperature for at least 4 h followed by incubation in 30% sucrose in PBS at room temperature for 24 h. The cryo-protected brains were sectioned at 40 μm on a cryostat microtome (Leica). After washing in PBST (0.3% Triton X-100 in PBS) for 5 min 3 times, brain sections were incubated in blocking solution (3% BSA, 0.3% Triton X-100 in PBS) at room temperature for 1 h. Then brain sections were incubated with the primary antibodies overnight at 4°C and immunofluorescence-tagged secondary antibodies at room temperature for 2 h. After staining, the brain sections were mounted on adhesion microscope slides (Genview) and encapsulated in sealed tablets containing 3 µg/ml DAPI (Solarbio, C0060).The following antibodies were used: anti-HA (1:500, 11867423001, Roche), anti-NeuN (1:500, ABN78, Milipore), anti-GFP (1:2000, ab13970, Abcam), AlexaFluor-555 conjugated goat-anti-rat (1:500, 4417, CST), AlexaFluor-488 conjugated goat-anti-rabbit (1:500, 111-545-003, Jackson ImmunoResearch Laboratories), and AlexaFluor-488 conjugated goat-anti-chicken (1:500, ab150169, Abcam).

For Immunofluorescence staining, paraffin-embedded tissue sections were subjected to a heat-mediated antigen retrieval procedure. To be specific, after sections were dried, dewaxed, and hydrated, they were heated to repair antigens. Antigens repair was performed with citric acid buffer (PH6.0) in a 92 °C water bath for 60 min and then restored at room temperature. Following that, tissue sections were incubated overnight with a primary antibody [(Col-2 pAb, #28,459–1-AP, Proteintech, Wuhan, China), (Sox-9 Rabbit mAb, #82,630, Cell Signaling Technology, MA, USA)] at 4 °C. The secondary antibody was then added to the sections, which were incubated for 60 min. The nucleus was counterstained with 75% DAPI (C0060, Solarbio) for 10 min. Finally, anti-fluorescence quencher was applied to seal the sections.
Cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and then blocked with 3% BSA (A8010, Solarbio). The primary antibodies were then incubated overnight in PBS at 4˚C, followed by an hour of incubation with the secondary antibodies at room temperature. The rest of the steps are the same as above.

HLE B-3 cells and BMs grown on 24-well plates following various treatments were fixed with 4% paraformaldehyde solution containing 0.18% Triton X-100 for 30 min at 4°C. After rinsing thrice PBS, specimens were incubated with PBS containing 2% goat serum (Shang hai yuan mu, Shanghai, China) for 1 h at 37°C. Fixed cells or BMs were then incubated for 1 h at 37°C with the rabbit pAbs against p21 (1:500), LMα4 (1:100), LMβ3 (1:50) and MMP-9 (1:100; 10375-2-AP, Proteintech). After washing thrice with PBS, cells or BMs were incubated with FITC-anti-rabbit IgG (1:250, A11008, Life Technologies, New York, USA) for 30 min at room temperature. Next, the cells were washed thrice with PBS and incubated with DAPI (1:5000, C0060, Solarbio, Beijing, China) in PBS for 2 min at room temperature. However, this step was not performed for BMs. Finally, the cells or BMs were visualized using a Leica DMRA immunofluorescence microscope and Leica software (Leica Microsystems, Wetzlar, Germany).

Treated BCP1 cells (1.5x10⁴) were seeded on polylysine-coated glass coverslips (WHB, 12-CS-LC) for 30 min. Cells on coverslips were fixed with 100% methanol for 30 min at room temperature and then washed with PBST for three times. Fixed cells were then permeabilized with 1% Triton X-100 in PBST for 15 min at room temperature and subsequently blocked with 1% BSA in PBST for 30 min at 37°C. Cells were incubated with a mouse anti-ORFK8 monoclonal antibody (Santa Cruz, F33P1) at a 1:100 dilution at 4°C overnight. The cells were then washed three times with PBST followed by incubation with an Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G secondary antibody for 60 min at 37°C. The cells were again washed with PBST three times and stained with DAPI (Solarbio, C0060) for 5 min at room temperature, and washed with PBST three times. The coverslips were mounted onto slides using an anti-quenching mounting buffer (Solarbio, S2100). Cells were imaged with a fluorescent microscope (Zeiss, M2).

Paraffin-embedded tissue sections were stained using ZO-1 antibody (1:100, ab221547, Abcam, UK), Claudin-1 antibody (1:1000, ab211737, Abcam, UK), and DAPI (C0060, Solarbio, China) for immunofluorescence. Two investigators, blinded to the treatment, independently evaluated the slides. SlideViewer 2.5.0 (DHISTECH Ltd., Hungary) was used for imaging, and Image J software (National Institutes of Health, Bethesda, Maryland) was used to analyze the fluorescence signal intensity.

Aliquots of 5 × 10³ MSCs labeled with CFSE and 5 × 10³ MEFs (or 1 × 10⁴ MEFs alone) in serum-free DMEM culture media were cultured on the upper layer of Corning cell culture inserts with an 8.0 μm polycarbonate membrane. A DMEM culture medium with 10% FBS and the indicated cytokines or chemicals was added below the cell’s permeable membrane. Following an incubation period of 24 h at 37 °C and 5% CO₂, the cells that had migrated through the membrane were harvested. For immunofluorescence, cells were fixed with 4% paraformaldehyde (Sigma) for 20 min. Then, 10% bovine albumin (BSA) was used to block the cells for 20 min at room temperature. Afterwards, the cells were incubated with anti-α-Sma (Servicebio, GB111364) overnight at 4 °C. On the second day, the cells were incubated with the TRITC-conjugated anti-rabbit antibody (Abclonal, AS040) for one hour at room temperature. DAPI (Solarbio, C0060) was used to stain the nucleus. Images were taken using a Zessis Axio Scope A1 FL for FISH microscope. For crystal violet staining, cells were fixed with methanol for 20 min and 0.1% crystal violet was added. Photos were taken using an Olymbus IX50/70 microscope system.

For tissue sections, the above protocol for Masson staining was followed before incubation with primary antibodies. For cell samples, the cells were fixed with 4% paraformaldehyde (Sigma) for 20 min. Then, the cells were permeated with 0.2% Triton X-100 for 20 min at room temperature. BSA (10%) was used to block the cells for 20 min at room temperature. After, the cells were incubated with primary antibodies overnight at 4 °C. On the second day, the cells and sections were incubated with DAPI (Solarbio, C0060) and TRITC- or FITC-conjugated secondary antibodies for one hour at room temperature. Images were taken using a Zessis LSM 800 laser scanning confocal microscope.