Rabbit anti mouse phospho stat3 tyr705

H&E and Alcian blue (AB) stains were performed on lung sections as described [35] (link). For IHC, monoclonal antibody against eosinophil specific major basic protein (anti-MBP, a kind gift from Drs. Nancy and James J. Lee, Mayo Clinic, Scottsdale, AZ) was used (1∶500) for eosinophils and anti-IL-22 antibody was used to detect IL-22 (1∶180). ABC staining kits were used to amplify the signal (Santa Cruz Biotechnology). Immunofluorescence of p-STAT3 was performed with rabbit anti-mouse phospho-Stat3 (Tyr705) (Cell Signaling, Danvers, MA) and Alexa Fluor 488-labeled donkey anti-rabbit IgG as secondary antibody (Invitrogen) and DAPI for nuclei. Tissue sections were mounted and examined using Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss) at 350 nm to assess p-STAT3 and 405 nm to assess cell nuclei.

For western blotting analysis, colon tissues were collected and homogenized with a RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA). After separation with SDS/polyacrylamide gel electrophoresis, proteins were transferred onto nylon membranes and then reacted with rabbit anti-mouse STAT3 and rabbit anti-mouse Phospho-STAT3 (Tyr705) antibodies (Cell Signaling Technology, Beverly, MA). Anti-β-actin antibody (Cell Signaling Technology) was used to confirm that equal amounts of proteins had been used for analysis. Signals were detected by using an LAS-4000 (GE Healthcare Japan, Hino, Japan).