Anti lotus tetragonolobus lectin

Frozen sections of kidneys were fixed with 4% paraformalin‐fixing solution for 15 min at room temperature. NRK‐49F cells cultured on coverslips were fixed with cold methanol:acetone (1:1) for 15 min at room temperature, followed by blocking with 10% normal donkey serum in PBS. Slides were incubated with primary antibodies against anti‐N‐OPN (Abcam, Cat. ab181440, 1:50), anti‐OPN (Boster Biotechnology, Cat. PB0589, 1:50), anti‐CD63 (Abcam, Cat. ab59479, 1:50), anti‐Lotus Tetragonolobus Lectin (LTL) (VECTOR Laboratories, Cat. FL‐1321,1:400), anti‐Peanut Agglutinin (PNA) (VECTOR Laboratories, Cat. FL‐107, 1:400), anti‐Dolichos Biflorus Agglutinin (DBA) (VECTOR Laboratories, Cat. FL1031, 1:400), anti‐Collagen I (Boster Biotechnology, Cat. BA0325, 1:50), anti‐Vimentin (Abcam, Cat. ab8978, 1:50), anti‐fibronectin (Sigma, Cat. F3648; 1:100), anti‐CD44 (Boster Biotechnology, Cat. M00052‐3, 1:50) and anti‐β‐catenin (BD Biosciences, Cat. 610154, 1:150). After washing with TBS‐T, slides were incubated with Cy2 or Cy3‐conjugated donkey anti‐ or anti‐rabbit IgG (Jackson Immuno‐Research Laboratories, West Grove, PA). Nuclei were stained with DAPI (Beyotime, Cat. C1006) according to the manufacturer's instructions. All images were taken by confocal microscopy (Leica TCS SP2 AOBS, Leica Microsystems, Buffalo Grove, IL).

P60 Het and WT kidney tissues stored in 3% glutaraldehyde were processed and embedded by the Department of Pathology, Tulane University. Ultimately, 60 nm sections were cut and imaged using a Hitachi H‐7100 electron microscope. Glomerular number was counted in Het and WT kidneys on P60 from 3 consecutive H&E‐stained sections/kidney adjacent to the longitudinal midplane (n = 3 kidneys/genotype). E13.5‐E15.5 4‐μm kidney sections from Mut and WT mice were processed for immunofluorescence using anti‐amphiphysin (ProteinTech, 1:200), anti‐active β‐catenin (ABC, Millipore, 1:400, anti‐Lotus Tetragonolobus Lectin (LTL) (1:400, Vector Laboratories), and anti‐cytokeratin (1:200, Sigma) antibodies. Immunostaining was performed by the immunoperoxidase technique with Vectastain Elite kit (Vector Laboratories, Burlingame, CA). Secondary antibodies were detected with Alexa Fluor dyes (Invitrogen). Specificity of immunostaining was documented by the omission of the primary antibody.