Anti egfp

All of the protein extracts were heated at 99°C for 5 minutes in SDS-PAGE solubilising buffer (58 mmol/L Tris HCl, 10% glycerol, 2% SDS, 0.004% bromophenol blue, pH 6.8) containing 7.5% dithiothreitol. The proteins were separated by means of SDS-PAGE electrophoresis on a 10% polyacrylamide gel, and transferred to a PVDF membrane. After blocking, the membrane was incubated with anti-ICln [5] (link), anti-actin I-19 (Santa Cruz Biotechnology, Dallas, Texas, USA), anti 4.1R C-16 (Santa Cruz Biotechnology) or anti-4.1R EPB41 (Sigma-Aldrich), anti-EGFP (Clontech), monoclonal anti-GAPDH (clone GAPDH-71.1, Sigma-Aldrich), anti-pan cadherin ABT35 (Abcam plc, Cambridge, UK), or anti-FLAG M2 antibody (Sigma-Aldrich), diluted in the blocking buffer at 4°C overnight, followed by several washes, and then by the secondary HRP-conjugated antibody. The Immobilon ECL system (Millipore S.p.A., Vimodrone, Italy) was used for detection.
The PVDF membrane was always stained using the amido black staining procedure in order to assess the efficiency of protein transfer and verify equal loading.
The bands were densitometrically analysed using the ImageJ software.

Primary antibodies used for confocal microscopy were: anti-F-actin (TRITC-phalloidin—Sigma), anti-alpha tubulin (clone DM1A, Sigma-aldrich), anti-pTyr-AF647 (clone PY99, Santa Cruz Biotechnology), anti-IFN-gamma (clone AN-18, BD Biosciences). Secondary antibodies used for confocal microscopy were: anti-rat AF568 (Molecular Probes) and anti-mouse AF594 (Molecular Probes). Antibodies used for flow cytometry were: anti-TCR DO11.10 PE (clone KJ1.26, MBL-medical and biological lab), anti-CD4 AF405 (clone RM4-5, Invitrogen), anti-CD80 PB (clone 16-10A1, BioLegend), anti-CD86 PE-Cy7 (clone PO3, BioLegend), anti-CD54 (ICAM1) APC (clone YN1/1.7.4, BioLegend), anti-CD40 PE-Cy7 (clone 3/23, BioLegend), anti-CD48 APC (clone HM48-1, BioLegend), anti-MHC-II PB (clone M5/114.15.2, BioLegend), anti-CD19 APC-H7 (clone 1D3, BD Biosciences), anti-CD21 APC (clone 7G6, BD Biosciences), anti-CD23 PE (clone B3B4, BD Biosciences), anti-CD69 FITC (clone H1.2F3, BioLegend), anti-CD95 PE (clone Jo2, BD Biosciences), anti-IFN-γ PE (clone XMG1.2, BD Biosciences). Primary antibodies used for western blot were: anti-EGFP (Clontech) and anti-actin (polyclonal, Sigma-Aldrich). Secondary antibodies used for western blot were anti-rabbit (GE Healthcare) and anti-mouse (Jackson Immunoresearch) antibodies, conjugated with horse radish peroxidase (HRP).

Paraffin sections were deparaffinized and rehydrated. Antigen retrieval was performed by microwave heating in 1 mM EDTA, pH 6.0 for 10 min. The following solutions were applied for blocking: Avidin/Biotin solutions (Vector Laboratories, Burlingame, CA) for 20 minutes and background buster (Accurate Chemical & Scientific, Westbury, NY) for 30 minutes. The following primary antibodies were applied overnight to detect the reporters: Anti-RFP (tdTomato reporter) (1:100, Rockland Inc. Limerick, PA) and anti-EGFP (podocyte specific reporter) (1:100, Clontech, Mountain View, CA). This was followed by the appropriate biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) and then by Streptavidin, AlexaFluor 488 conjugate (1:200, Life Technologies - Molecular Probes, Grand Island, NY). Slides were mounted with Vectashield/DAPI mounting media (Vector Laboratories, Burlingame, CA). Fluorescent images were collected at 400x magnification. Coverslips were gently removed by soaking slides in PBS, and slides were subsequently stained for p57/PAS as described above. Brightfield images were collected and combined with immunofluorescent images, showing the same glomeruli in both sets of images.