Alexa fluor 488 goat anti mouse igg antibody

After HUVECs grown on glass slides were treated with 4% paraformaldehyde, the slides were incubated first with 0.1 mol/L glycine in phosphate-buffered saline (PBS) and then with 0.1% Triton X-100 in PBS. The slides were washed and then incubated with 5% swine serum in PBS for 1 hour.
For nuclear factor kappa-B (NF-κB) subunit p65 immunostaining, slides were incubated with diluted mouse anti-NF-κB p65 antibody (Santa Cruz Biotechnology, Dallas, TX, USA). After washing with PBS three times, the slides were incubated with Alexa Fluor 488 goat anti-mouse IgG antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 hour. Then, the slides were washed and mounted in Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Images were captured with a fluorescence microscope.

Colon tissue samples from three groups (vehicle, TCDD, and TCDD+Sch527123) were fixed in 4% paraformaldehyde diluted in PBS overnight. Fixed colons were sectioned (5 µm thick) and placed on coated slides. Slides were incubated for 30 min in glycine 0.1% and 1× Triton for tissue permeabilization for Arg1 staining or with glycine 0.1% only for CD11b and Gr-1 staining. Slides were then incubated with primary mouse anti-Arg1 antibodies or primary rat anti-CD11b and anti-Gr-1 antibodies purchased from Cell Signaling (Danvers, MA, USA) at 4 °C overnight, followed by a 2 h incubation at room temperature with secondary Alexa Fluor 488 goat anti-mouse IgG antibody for Arg1, Alexa Fluor 488 goat anti-rat IgG for CD11b, and Cy5 goat anti-rat IgG antibody for Gr-1. Fluorescent imaging of colon sections was taken using a Leica DM 2500 optical microscope from Leica Microsystems (Buffalo Grove, IL, USA). The quantification of cell markers was calculated as corrected total cell fluorescence (CTCF) using Image J software (National Institutes of Health and the Laboratory for Optical and Computational Instrumentation).