Ab175473

After 4% PFA fixation, cells were blocked by 5% bovine serum albumin (BSA, A7906, Sigma) in DPBS containing 0.1% Triton X-100 for 1 h, followed by the incubation of primary antibodies against integrin beta 1 (12G10, ab30349, Abcam, 1:200), paxillin (Y113, ab32048, Abcam, 1:200), CD49c (integrin alpha 3, ASC-1, MA5-28565, Invitrogen, 1:50), talin 1 (8D4, ab157808, Abcam, 1:100), vinculin (EPR8185, ab129002, Abcam, 1:100), FAK (#3285, Cell signaling Technology, 1:200), α-actinin (H-2, sc-17829, Santa Cruz Biotechnology, 1:200), and Phospho-Myosin Light Chain 2 (Thr18/Ser19) (pMLC, #3674, Cell Signaling Technology, 1:100) in 1% BSA for overnight at 4 °C. The samples were washed twice with PBS before applying fluorescence-conjugated secondary antibodies, including Goat Anti-Mouse lgG H&L (Alexa Fluor® 488) (ab150113, Abcam, 1:1000), Goat Anti-Rabbit lgG H&L (Alexa Fluor® 488) (ab150077, Abcam, 1:1000), Goat Anti-Rabbit lgG H&L (Alexa Fluor® 568) (ab175471, Abcam, 1:1000), Donkey Anti-Rabbit lgG H&L (Alexa Fluor® 647) (ab150075, Abcam, 1:1000), Goat Anti-Mouse lgG H&L (Alexa Fluor® 568) (ab175473, Abcam, 1:1000), Goat Anti-Mouse lgG H&L (Alexa Fluor® 647) (ab150115, Abcam, 1:1000), in 1% BSA with or without dye-conjugated phalloidin and DAPI (R37606, Invitrogen) at room temperature for 45 min.

Cells were seeded on a 24-well plate at 1 × 10⁵ cells/well. After 2 days, cells were fixed using pre-chilled 4% paraformaldehyde (C104190, Aladdin Biochemical Technology, Shanghai, China) for 20 min. Then, 0.1% of TritonX-100 (T109026, Aladdin) was added for incubation for 15 min to increase the permeability. Subsequently, cells were incubated in 1% bovine serum albumin (BSA) for 30 min for blockade of any non-specific binding sites. Next, an incubation lasted for 1 h with the use of primary antibodies against Cytokeratin (ab52625, 1:100, Abcam, ZO-1 (ab61357, 1:250, Abcam), and Vimentin (ab8978, 1:100, Abcam). Afterward, secondary antibody Alexa Fluor 568-conjugated goat anti-mouse immunoglobulin G (IgG) (ab175473, 1: 500, Abcam) or AlexaFluor 488-conjugated goat anti-rabbit IgG (ab150077, 1:200, Abcam) was added for incubation for 1 h. Next, Hoechst 33342 (1:10,000; 1 mg/mL) was added for nuclear staining. The stained cells were imaged under a fluorescence microscope.

Kidney biopsies and 293 T cells fixed in neutral buffered formalin were embedded in paraffin or optimal cutting temperature compound by using standard procedures. Frozen and paraffin sections were stained with immunofluorescence, respectively. Immunofluorescent staining and images were obtained by a Nikon A1R Meta confocal microscope. Cover slips were observed.
The antibodies used were list below: anti-Cubilin-C-terminal antibody (1:500, ab191073, Abcam), Rat Cubilin(CUBN) polyclonal antibody (1:100, 31010, Bicell Scientific), anti-Synaptop-odin antibody (1:50, 21064-1-AP, Proteintech), anti-Wilms Tumor Protein antibody (1:50, ab89901, Abcam), anti-COL4A3 antibody (1:100, Kingmed, Guangzhou, China), anti-COL4A5 antibody (1:100, Kingmed, Guangzhou, China), anti-Amnionless antibody (1:10, sc-365384, Santa Cruz), anti-Megalin Antibody (1:30, CD7D5, Novus Biologicals), goat polyclonal secondary antibody to mouse Alexa fluor 488 (1:400, ab150113, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 555 (1:400, ab150078, Abcam), rabbit monoclonal to HA tag (1:500, ab236632, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 647 (1: 400, ab150079, Abcam), goat anti-mouse Alexa fluor 568 (1: 400, ab175473, Abcam), DAPI (1:1000, C1002, Beyotime).

After ADSC differentiation, the obtained cells were fixed with 4% formaldehyde for 15 min. This was followed by permeabilization with 0.5% Triton X solution (93443, Sigma-Aldrich, USA). The proteins osteocalcin and RUNX2 were detected after incubation of the samples with anti-osteocalcin monoclonal antibody OCG3 (1:500, ab13420, Abcam, UK) and anti-RUNX2 monoclonal antibody 2B9 (1:50, ab76956, Abcam, UK) primary antibodies, respectively. Subsequently, one of two fluorochrome-conjugated secondary antibodies was used: Alexa Fluor 488 (1:1000, ab150113, Abcam, UK) or Alexa Fluor 568 (1:1000, ab175473, Abcam, UK). Cell nuclei were stained with DAPI dye (VECTASHIELD Vibrance Antifade Mounting Medium, H-1700, Vector, USA). The isotype controls were Mouse IgG2a (ab18415, Abcam, UK) and IgG3 (ab18392, Abcam, UK).

Fixed organoids are embedded in paraffin and sectioned by HKU histopathology service by Department of Pathology. Paraffin sections are dewaxed and rehydrated by going through a ladder of xylene, ethanol and distilled water as described. Sections are then blocked and permeabilized with section staining buffer (5% FBS/0.2% gelatin/0.25% Triton X 100/PBS) for 30 min. Primary antibodies of anti-SARS/SARS-CoV-2 Nucleocapsid Monoclonal Antibody (Invitrogen, MA1-7404) is diluted in section staining buffer at 1:400 and sections are stained at 4 °C overnight. Sections are then washed with section staining buffer 3 times for 15 min each. Sections are then stained with secondary antibodies (Abcam, AB175473) diluted at 1:800 and 0.25 µM DAPI at 4 °C overnight. After three rounds of washing, direct conjugated antibodies are stained at 1:400 dilution at 4 °C overnight. Slides are washed three times with staining buffer before mounting in 70% glycerol/PBS and sealed with nail polish.

CH were seeded on 1 cm diameter glass coverslips at a density of 10⁴ cells/cm². At confluence, cells were treated with 10 ng/mL TNFα, 5 ng/mL TGF-β1 or a combination of the two treatments. After 3 days, samples were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and incubated overnight at 4 °C with the antibody raised against Cx43 C-terminal (#3512, Cell Signaling, Danvers, MA, USA, 1:100 diluted). Specific binding was revealed with a secondary antibody conjugated to AlexaFluor 488 (A-11001, Invitrogen, Waltham, MA, USA, 1:2000 diluted), and coverslips were mounted with ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA, USA). Samples were analyzed by wide-field fluorescence microscopy (BX51, Olympus). For confocal imaging, a double staining for Cx43 (primary antibody #3512, Cell Signaling, Danvers, MA, USA, 1:100 diluted and secondary antibody conjugated to AlexaFluor 488, Ab150073 Abcam, Cambridge, UK, 1:1000 diluted) and β tubulin (primary antibody T7815 1:500 diluted and secondary antibody conjugated to AlexaFluor 488, Ab175473 Abcam, Cambridge, UK, 1:1000 diluted) was performed following standard procedures. Samples were analyzed by the confocal laser scanning microscope TCS SP8 (Leica Microsystems CMS GmbH, Wetzlar, Germany). Images were acquired with a 63× objective and analyzed using Fiji software (ImageJ 1.51).