Camedia c 2000 digital camera

The transverse colon was embedded in Tissue-Tek (Sakura, Tokyo, Japan), snap-frozen in isopentane, cooled in liquid nitrogen vapor, and stored at −70 °C. Then, 5-μm acetone-fixed cryosections were cut on a cryostat CM 1860 UV (Leica Microsystems, Wetzlar, Germany) and put on SuperFrost/Plus slides (Thermo Fisher Scientific, Darmstadt, Germany) and were kept at −40 °C until labeling. The sections were incubated with 10% normal rabbit serum (Life Technologies, Carlsbad, CA, USA) in a humid chamber for one h at RT. Labeling by anti-TLR4 rabbit polyclonal antibodies (Novus Biologicals, Centennial, CO, USA) was performed overnight at 4 °C. The sections were incubated with secondary antibody, peroxidase-conjugated F(ab’)2-goat anti-rabbit IgG (H+L) (Life Technologies, Carlsbad, CA, USA) for 2 h at RT. The TLR4 localization was visualized by an incubation with AEC substrate kit (Sigma-Aldrich, St. Louis, MO, USA) and examined under an Olympus BX 40 microscope with Olympus Camedia C-2000 digital camera (Olympus, Tokyo, Japan). Control sections without primary antibody were treated in the same way.

Acid mucin-producing cell density per area of the tunica mucosa was assessed as described elsewhere [61 (link)]. Briefly, Carnoy’s fluid-fixed terminal ileum and colon were dehydrated and embedded in paraffin, and 5 μm cross-sections were stained with Alcian Blue and post-stained with Nuclear Fast Red. The specimens were examined under an Olympus BX 40 microscope with an Olympus Camedia C-2000 digital camera (Olympus, Tokyo, Japan).

The prepared 5 μm cryosections were incubated with 3% H₂O₂ for 10 min and blocked with 10% normal rabbit serum (RAGE; Life Technologies, Carlsbad, CA, USA) or 10% normal goat serum (TLR4; Life Technologies) for 1 h at RT. Anti-human RAGE goat polyclonal antibodies (Bio-Rad, Hercules, CA, USA) or anti-human TLR4 rabbit polyclonal antibodies (Novus Biologicals, Centennial, CO, USA) labeled the sections overnight at 4 °C. After, they were incubated with a secondary antibody, peroxidase-conjugated F(ab)₂ rabbit anti-goat IgG (RAGE; Jackson ImmunoResearch Europe, Ely, UK) or goat anti-rabbit IgG (TLR4; Life Technologies, Carlsbad, CA, USA) for 2 h at RT. The receptors were visualized by AEC substrate (Sigma-Aldrich, St. Louis, MO, USA) and nuclei were counterstained with Mayer’s hematoxylin (Diapath, Martinengo, Italy). The preparates were examined under an Olympus BX 40 microscope with Olympus Camedia C-2000 digital camera (Olympus, Tokyo, Japan). Control sections without primary antibodies were treated in the same way [35 (link)].