rabbit anti nephrin primary antibody  (ProSci Incorporated)

Journal: Nephrology Dialysis Transplantation

Article Title: Inhibition of the glucocorticoid receptor attenuates proteinuric kidney diseases in multiple species

doi: 10.1093/ndt/gfad254

Figure Lengend Snippet: Pharmacological inhibition in zebrafish model. ( A ) Schematic of the zebrafish model. Zebrafish larvae (age 6 days post-fertilization) have a single glomerulus (in red) fused to a pair of tubules. If podocytes are partially depleted upon treatment with the prodrug MTZ, zebrafish larvae become proteinuric and display pericardial edema formation. ( B ) Timeline of the experiment. Animals were treated for 5 h with dexamethasone or mifepristone prior to addition of MTZ (i.e. induction of podocyte damage). ( C ) Edema formation in zebrafish larvae exposed to MTZ. (C1, C1′) Images of larvae with and without edema. Edematous larvae exhibit pericardial, yolk sac (arrows in C1 and C1′) and periorbital edema. (C2) Larvae with edema show decreased gc-eGFP fluorescence in the vasculature (arrows indicating segmental tail vessels in C2 and C2′), indicating a leaky filtration barrier, and decrease of mCherry fluorescence in the glomerulus (arrows in C3 and C3′). Scale bar: 100 µm. ( D ) Analysis of edema formation, a parameter of glomerular permeability defect, in mifepristone-, dexamethasone- and vehicle-treated zebrafish larvae. Edema formation is reduced by glucocorticoid agonism and antagonism. ( E ) Confocal laser scanning micrographs of glomeruli show decreased staining for nephrin (green) exclusively in larvae with edema compared with healthy control larvae (scale bar represents 10 µm). ( F ) Mean values of qRT-PCR for nphs2 mRNA levels normalized to 18 s rRNA and compared with DMSO controls of whole larval lysates are shown. A 48-h exposure to 100 µM MTZ reduced nphs2 mRNA levels to 0.06. Co-treatment with 100 nM dexamethasone or with 100 nM mifepristone resulted in an elevated nphs2 expression (0.31 ± 0.05 and 0.25 ± 0.11, respectively), compared with control. The results of three independent experiments ( n = 20 larvae per experiment) are expressed as mean mRNA levels ± SD. * P < .05, by one-way ANOVA followed by Bonferroni's post hoc test. Data represent means ± SD.

Article Snippet: After blocking [2% fetal bovine serum (FBS), 2% BSA, 0.2% fish gelatin in 1× PBS] for 1 h, the sections were incubated with a polyclonal rabbit anti-zebrafish nephrin primary antibody (1/2000, Innovagen, Lund, Sweden) at 4°C overnight.

Techniques: Inhibition, Fluorescence, Filtration, Permeability, Staining, Control, Quantitative RT-PCR, Expressing