Rabbit anti gfp

Flushed intestines were fixed in 4% paraformaldehyde, dehydrated and then embedded in paraffin. Slide-mounted sections were prepared with a rotary microtome. Immunostaining was performed according to standard protocols. The sections were deparaffinised in xylene and rehydrated using a descending alcohol series. Antigen retrieval was performed by incubating the sections in 10 mM sodium citrate, pH 6.5, in a heated water bath at 97 °C for 40 min. Following a further one-hour incubation at room temperature in 0.4% Triton X-100 in PBS, the sections were transferred to a blocking solution (BSA 1% in PBS buffer). Anti-GFP antibodies were used to reduce green autofluorescence emitted from the tissue and allow detection of GFP-expressing VE14821. Sections were incubated overnight at 4 °C with primary antibodies: mouse anti-claudin 1 (Invitrogen, diluted 1:100) and rabbit anti-GFP (Roche, diluted 1:200). Fluorescence-labelled anti-mouse DyLight 488 (Thermo Fisher Scientific, diluted 1:500) and anti-rabbit A568 (Thermo Fisher Scientific, diluted 1:2000) were used as secondary antibodies. Nuclei were stained with Hoechst 33342 (Sigma). Sections were scanned with a digital slide scanner (Pannoramic Scan 3DHistech).