F3648

Kidneys were fixed in 10% formalin and embedded in paraffin. For detection of Sirius red-positive collagen, deparaffinized kidney sections (4 μm) were rehydrated, predifferentiated with 0.2% phosphomolybdic acid for 5 min, stained with 0.1% picrosirius red (Direct Red 80, REF: 365548; Sigma-Aldrich, St. Louis, MO, USA) in picric acid for 90 min and differentiated with saturated picric acid. Primary antibodies against fibronectin (1:4800, F3648, Sigma), collagen type IV (1:500, ab19808, Abcam, Cambridge, UK), endomucin (1:20,000, 14-5851-82, EBioScience, San Diego, CA), VCAM-1 (1:800, 553330, BD Biosciences, Breda, The Netherlands), F4/80 (1:100; kindly provided by the Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands) and CD206 (1:2000, ab64693, Abcam) were used for immunohistochemistry (IHC) of paraffin-embedded tissues. Frozen sections were fixed with acetone/ethanol and immunostained with primary antibodies against VSV-G (1:2000, V4888, Sigma-Aldrich), luciferase (1:3000, ab181640, Abcam), FLT-1 (1:9000, AF471, R&D Systems) and VCAM-1 (1:800, 553330, BD Biosciences). The appropriate Envision (Dako), Impress (Vector Laboratories) or Goat-on-Rodent (BioCare) HRP-conjugated secondary reagents were used with DAB+ as the chromogen. Non-specific isotype-matched antibodies were used as a negative control.

To confirm the presence of individual proteins in the isolates, proteins eluted with Gly-HCl buffer and 0.2 M GalNAc in acetate buffer were pooled, separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Blots were incubated with the following antibodies: polyclonal rabbit anti-semenogelin 1 (1:1000, MBS968887, MyBioSource, San Diego, CA, USA), anti-semenogelin 2 (1:1000, MBS9132638, MyBioSource, San Diego, CA, USA) anti-lactotransferrin (1:1000, ab197811, Abcam, Cambridge, UK), anti-prolactin-inducible protein (1:600, ab198018, Abcam, Cambridge, UK), anti-fibronectin (1:2000, F3648, Sigma–Aldrich, St. Louis, MO, USA) and next with horseradish peroxidase labeled goat anti-rabbit IgG (1:5000, 111-035-045, Jackson Antibodies, Suffolk, UK). For HRP staining diaminobenzidine (0.1 mg/mL) and H₂O₂ (0.04%) in 0.1 M citric buffer, pH 5.5, were applied.

Immunohistochemistry was performed on formalin-fixed, paraffin-embedded kidney sections from human and mouse using antibodies against RXRα (ET7108-99, Huabio, 1:200 dilution), α-SMA (ab5694, Abcam, 1:100 dilution), Collagne-1 (ab34710, Abcam, 1:800 dilution), and Fibronectin (F3648, Sigma, 1:1000 dilution). Briefly, deparaffinized and rehydrated sections were boiled in citrate antigen retrieval solution for antigen retrieval. After incubation with 10% goat serum, sections were incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (ZLI-9018, ZSGB-BIO, 1:20 dilution). DAB-positive staining was assessed by two experienced pathologists in a blinded manner and evaluated by H score as described previously^{40 (link)}. For immunofluorescence, deparaffinized and rehydrated sections were blocked and incubated with antibodies against KIM-1 (AF1817, R&D System, 1:200 dilution) overnight at 4 °C, followed by incubation with Alexa Fluor 555-conjugated secondary antibodies (Invitrogen, 2273776, 1:1000 dilution) and fluorescein-LTL (Vector Lab, FL-1321, 1:1000 dilution). The tissue slides were imaged with a fluorescence microscope (DMi8, Leica).

Embryos for most antibodies were fixed at the indicated stage in 4% paraformaldehyde, permeabilized in PBST (PBS+0.5% Triton X-100), and blocked in PBST+2% bovine serum albumin. Antibodies and concentrations were as follows: anti-Pax2a (GTX128127, Genetex; 1:200); anti-pSmad3 (ab52903, Abcam; 1:200); anti-Laminin 1 (L9393, Sigma-Aldrich; 1:100); anti-fibronectin (F3648, Sigma-Aldrich; 1:100); anti-GFP (A10262, Invitrogen; 1:200).
In accordance with methods described by Carrara et al. (2019) (link), anti-Nidogen 1/Entactin (ab14511, Abcam) was used at 1:100 and required a modified embryo preparation after 4% paraformaldehyde fixation: embryos were permeabilized in 30 μg/ml Proteinase K for 15 min, and blocked in 0.8% PBST+10% sheep serum+1% bovine serum albumin. The antibody was applied in 0.8% PBST+1% sheep serum+1% bovine serum albumin.
Secondary antibodies used were: Alexa Fluor 488 goat anti-mouse (A-11001, Invitrogen), Alexa Fluor 488 goat anti-rabbit (A-11008, Invitrogen), Alexa Fluor 488 goat anti-chicken (A-11039, Invitrogen), all used at 1:200. Nuclei were detected by incubation with 1 µM TO-PRO-3 iodide (T3605, Invitrogen). Embryos were cleared through a series of 30%/50%/70% glycerol (in PBS) prior to imaging.

Western blot was performed using a lysate of kidney cortex or cultured PTECs. The primary antibodies used were as follows: anti-CPT1α (ab128568, Abcam), anti-KIM1 (ab47635, Abcam), anti-NGAL (ab63929, Abcam), anti-E-cadherin (610181, BD Company), anti-AQP1 (ab168387, Abcam), anti-vimentin (ab92547, Abcam), anti-fibronectin (F3648, Sigma Aldrich), anti-collagen I (1310-01, Southern Biotech) and anti-Tubulin (T6074, Sigma Aldrich). Western blot was performed three times independently. Quantification was completed by scanning and analyzing the intensity of hybridization signals by using NIH Image program.