Goat anti mouse igg af488

Sections cut from OCT-embedded fresh tissues (5-12µm thickness) were fixed for 20 minutes in Cytofix/Cytoperm (BD) at room temperature (RT), washed in PBS and blocked using 10% human plasma, 10% normal donkey serum and 10% normal goat serum in PBS containing 1% bovine serum albumin (BSA) for 30 minutes at RT. 5µg/mL purified mouse anti-CD3 (UCHT1; BioLegend) and 0.67µg/mL rabbit anti-CD31 (polyclonal; Bethyl Labs #IHC-00055) antibodies were incubated with tissue sections for 12 hours at 4°C. Following washing, tissue sections were incubated with goat anti-mouse IgG-AF488 (Abcam) and donkey anti-rabbit IgG-AF555 (Abcam) detection antibodies at a dilution of 1:500 for 1 hour at RT. Following washing, tissue sections were blocked using 10% normal mouse serum in PBS containing 1% BSA for 30 minutes at RT. Sections were then incubated with mouse anti-GD2-AF647 (14.G2A; BD) at a dilution of 1:100 for 2 hours at RT. After washing, sections were mounted using ProLong Gold anti-fade mounting medium with DAPI (Life Technologies), cured overnight, and sealed with nail varnish before viewing.

Biopsies from six active and four inactive CD patients were harvested during routine exploratory colonoscopy and immediately placed in complete cold RPMI medium for transport. They were fixed in 4% paraformaldehyde overnight then embedded and frozen in OCT. Six micrometre sections were cut, then rinsed in PBS three times to eliminate OCT. Tissue permeabilization was performed by incubating slides in 0.6% Triton for 5 min, then incubated in 2× Saline Sodium Citrate (SSC) buffer for 25 min. Slides were washed in DEPC‐treated water (10 dips), then in 0.065 M trietanolamine (TEA, Sigma; 10 dips), and finally twice in 2× SSC. Tissues were hybridized with 150 μlof 16S‐probes (50 pM final; Appendix Table S2) overnight, at room temperature. Slides were washed in successive baths of decreasing SSC concentrations (4×, 2×, 1×, 0.5×, 0.1×) then blocked for 1 h in a 1% BSA, 5% FBS, 0.3% Triton‐X100 PBS solution. All subsequent washing steps were carried out in PBS. Mouse anti‐human IgA2 antibodies (Southern Biotech, 9140–01) were deposited over each section at a 1/500 dilution and incubated for 2 h at room temperature. Slides were washed and secondary antibodies (goat anti‐mouse IgG‐AF488, abcam) were then added for 1 h. Slides were washed in PBS then distilled water, air‐dried, and sealed with Vectashield (Vectra, H1000) for confocal microscopy.