Ecl ap conjugated anti rabbit mouse igg

The colon tissue samples were lysed in a RIPA buffer containing a 2% phosphatase-inhibitor (Thermo Fisher Scientific, Waltham, MA, United States) and a 1% mammalian-protease inhibitor (Sigma-Aldrich). 50 μg of extracted proteins were run a single track of 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred electrophoretically onto cellulose membranes. The membranes were blocked in 5% non-fat dry milk for 60 min, and then incubated with primary antibodies against nNOS (1:1000; Cell Signaling, Boston, MA, United States), Sub-P (1:100; Novus biologicals, Centennial, CO, United States), ChAT (1:100), NGF (1:200), COX2 (1:300), Tryptase (1:500), PGP9.5 (1:1000) and GAPDH (1:10,000) (all Abcam, Cambridge, United Kingdom), and PGE2 polyclonal antibody (1 : 200; Bioss Inc., Woburn, MA, United States) (Chia et al., 2017 (link)) overnight at 4°C. The membranes were washed 3× with TBS-T (TBS mixed with 0.1% Tween-20) and then, they were incubated with ECL AP-conjugated anti-rabbit/mouse IgG (1:3,000; GE Healthcare, United Kingdom) for 90 min in room temperature. Quantitative western blot results were obtained by densitometric analysis using image processing and analysis in Java (Image J, NIH, Bethesda, MD, United States). The percent change of relative intensity was calculated against control samples.

Colon tissue samples were lysed in a RIPA buffer containing a 2% phosphatase-inhibitor (Thermo-Scientific, Waltham, MA) and a 1% mammalian-protease inhibitor (Sigma-Aldrich).
50 μg of extracted proteins were run a single track of 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the separated proteins were transferred electrophoretically onto cellulose membranes. The membranes were blocked in 5% non-fat dry milk for 60 min, and then incubated with primary antibodies against nNOS and p-Akt (both 1:1,000; Cell Signaling, Boston, MA); ChAT (1:100), GDNF (1:500) and GAPDH (1:10,000) (Abcam, Cambridge, UK) overnight at 4°C. The membranes were washed 3x with TBS-T (TBS mixed with 0.1% Tween-20) and then, they were incubated with ECL AP-conjugated anti-rabbit/mouse IgG (1:3,000; GE Healthcare, UK) for 90 min in room temperature. Quantitative analysis was done using Image-J software (NIH, Bethesda, MD).