Goat anti lysozyme c

Staining was essentially as described [36] (link). Cells on cover slips were fixed in 2% PFA at 4°C for 15–30 min, permeabilized with 0.1% Triton-100 (Fisher, Pittsburgh, PA) for 15 min, and blocked with 10% normal donkey serum (Jackson ImmunoLabs, West Grove, PA) in 2.0% BSA for 45 min-1 h before applying primary Ab at dilutions ranging from 1∶50-1∶500 for 1 hr at RT or overnight at 4°C using rabbit anti-IL-6 (Abcam 6672 or 7737, Cambridge, MA), goat anti-NFκB p50 (Santa Cruz; C-19), rabbit anti-NFκB p65 (Santa Cruz; C-20), goat anti-IκB (Santa Cruz), goat anti-lysozyme C (Santa Cruz; C-19), or sheep anti-rat albumin (Bethyl Laboratories, Inc., Montgomery, TX). After washing with 0.5% BSA, cells were incubated with secondary Abs ligated to either Alexa 488, Cy3, or Cy5 (Jackson ImmunoLabs, West Grove, PA), washed, stained with Hoechst to visualize nuclei for 30 s and mounted with gelvatol. Images were taken using epi-fluorescence connected to a digital CCD camera (Olympus Provis, Malvern, NY, USA) or by single slice confocal (Olympus Fluoview 1000). Actin was visualized using Alexa-647-conjugated phalloidin. In each experiment, all exposures were gated the same for comparative purposes. Photoshop CS3 (Adobe Systems, San Jose, CA) or Metamorph Offline version 7.5.4.0 (MDS Analytical Technologies, Sunnyvale, CA) were used for image analysis.

Samples from the ileum and colon were fixed in 10% neutral formalin. Paraffin sections were stained with hematoxylin and eosin (H&E), periodic acid-Schiff, or immunofluorescence by standard protocols. The antibodies used were rabbit anti-muc2 and goat anti-lysozyme C (Santa Cruz Biotechnology, Dallas, TX). After antigen retrieval with citrate buffer, immunostaining for MUC2 and lysozyme was performed by overnight staining with a 1:100 dilution of the primary antibody at 4°C, followed by staining with a 1:200 dilution of the secondary antibody conjugated with Alexa Fluor 488 (Life Technologies, Grand Island, NY). The slides were mounted with SlowFade gold antifade reagent (Life Technologies, Grand Island, NY) after counterstaining with 4′,6-diamidino-2-phenylindole (DAPI).