Alexa fluor conjugated secondary antibody

Cultured cells were fixed in 4% paraformaldehyde for 20 min, permeabilized in PBS containing 0.2% Triton X-100 and 3% bovine serum albumin, and incubated with primary antibodies to the indicated protein of interest, followed by Alexa Fluor–conjugated secondary antibodies (Jackson ImmunoResearch).
For human kidney biopsies and mouse kidney samples, 4-pm sections of paraffin-embedded kidneys were submitted to antigen retrieval protocols using high temperature (120°C) and high pressure in citrate buffer and a pressure cooker. Sections were then incubated with primary antibodies as indicated and appropriate Alexa Fluor-conjugated secondary antibodies (Jackson ImmunoResearch). Immunofluorescence for human tissue was performed on n = 10 kidney tissue per group of renal disease and n = 5 controls. The control group was composed of healthy renal peritumoral tissues. Renal function and albumin/creatinine ratio were determined at the time of biopsy.

Immunohistochemistry staining procedures have been previously described. ^{2 (link)} Tissue slides were incubated with antibodies against p- vascular endothelial-cadherin (p-VE-cadherin) (Cell Signaling, Danvers, MA), endothelial marker CD-31 (R&D Systems, Minneapolis, MN) and smooth muscle actin (SMA) (Sigma-aldrich, St Louis, MO). Slides were then incubated with the appropriate alexa fluorconjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA). A Nikon E800 Eclipse microscope (Nikon, Tokyo, Japan) was used to capture images at x20 magnification in five random fields from each animal (4 animals per group). Image J (Image Processing and Analysis in Jave) was used to analyze vascular density. Capillaries were defined as CD31 positive structures between 10-800 pixels and arteries were defined as smooth muscle actin positive structures with lumens greater than 800-infinity pixels.

Integral Molecular, Inc. (Philadelphia, PA) performed specificity testing of V_H-Fc ab8 using the Membrane Proteome Array (MPA) platform. The MPA comprises 5,300 different human membrane protein clones, each overexpressed in live cells from expression plasmids that are individually transfected in separate wells of a 384-well plate (Tucker et al., 2018 (link)). The entire library of plasmids is arrayed in duplicate in a matrix format and transfected into HEK-293T cells, followed by incubation for 36 h to allow protein expression. Before specificity testing, optimal antibody concentrations for screening were determined by using cells expressing positive (membrane-tethered Protein A) and negative (mock-transfected) binding controls, followed by flow cytometric detection with an Alexa Fluor-conjugated secondary antibody (Jackson ImmunoResearch Laboratories). Based on the assay setup results, V_H-Fc ab8 (20 μg/ml) was added to the MPA. Binding across the protein library was measured on an iQue3 (Ann Arbor, MI) using the same fluorescently labeled secondary antibody. To ensure data validity, each array plate contained positive (Fc-binding; SARS-CoV-2 S protein) and negative (empty vector) controls. Identified targets were confirmed in a second flow cytometric experiment by using serial dilutions of the test antibody. The identity of each target was also confirmed by sequencing.