Secondary goat antirabbit antibodies

Following a brief rinse in phosphate buffer 0.01 M, mice were perfused with 4% paraformaldehyde and 14% picric acid; brains were post‐fixed for at least 2 hr and then cryoprotected in a 50/50 mixture of fixative and 20% sucrose in 0.01 M phosphate‐buffered saline (PBS) for at least 24 hr. Sections were cut at 60 μm on a sledge microtome with a freezing stage (Yamato REM‐710 electrofreeze MC‐802A), washed in PBS and incubated in 20% normal goat serum. Primary antibodies to tyrosine hydroxylase (rabbit polyclonal 1:5,000, Enzo Life Sciences) or glial fibrillary acid protein (GFAP, rabbit polyclonal, Dako; 1:500) were incubated overnight at 4°C and stained with secondary goat antirabbit antibodies (Life Technologies; 1:200). At least 2 hr were allowed for binding before rinsing in PBS. Sections were mounted on slides; Vectamount AQ (Vector) or occasionally Santa Cruz mountant with DAPI was used to fix the coverslips. A spinning disk confocal microscope (Olympus BX‐DSU) was used and pictures were taken using Neurolucida software and a Hammatsu (EM‐CCD C91) camera.

Jejunal IEC from three-month-old wild-type and villin-Cre Hdac1^−/−; Hdac2^−/− mice were enriched by EDTA treatment. IEC were lysed in 1 X Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol) supplemented with protease and phosphatase inhibitors. Protein concentrations were determined with the Pierce BCA Protein Assay kit (Therrmo Fisher Scientific). Amounts of 15 µg of total proteins were separated on 4–12% SDS-polyacrylamide gels, and transferred on PVDF membranes (Roche Molecular Biochemicals). Membranes were incubated 1 h at room temperature or overnight at 4 °C with primary antibodies including rabbit anti-phospho-Stat3 (#9145, Cell Signaling), rabbit anti-Stat3 (#12640, Cell Signaling), rabbit anti-phospho-p38 (#4511, Cell Signaling), rabbit anti-claudin 3 (341700, Invitrogen), rabbit anti-cleaved Notch (#4147, Cell Signaling), rabbit anti-phospho-S6 (#4858, Cell Signaling) and GAPDH (#2118, Cell Signaling). Primary antibodies were recognized with secondary goat anti-rabbit antibodies (Life Technologies) before immune complex detection with Amersham ECL^TM Western blotting detection reagents (GE Healthcare, Mississauga, ON, Canada).