Histology, immunostaining, Alcian Blue-Periodic Acid Schiff (AB-PAS), histochemical staining, and fluorescence in situ hybridization

Colons were isolated, fixed, paraffin-embedded, and sectioned according to standard protocols. Intestine sections (5 μm) were processed for hematoxylin and eosin staining or for immunostaining. Immunohistochemistry was performed with R.T.U. vectastain kit (Vector Laboratories) with DAB substrate. Sections were counterstained lightly with Hematoxylin afterwards. For immunofluorescence staining, secondary antibodies used are goat anti-rabbit AlexaFluor 488 and donkey anti-rat AlexaFluor 594 (Invitrogen). Sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Primary antibodies used are: mouse anti-ki67 (BD Pharmingen, 550609), rat anti-CD4 (Ebioscience Inc, 14-9766-80), rat anti-Ly6G (BD Pharmingen, 551459), rabbit anti-F4/80 (Novus Biologicals Inc, NBP2-12506), rat anti-ZO-1 (Developmental Studies Hybridoma Bank, R26.4C), rabbit anti-hnRNPI (gift from Dr. Douglas Black), rabbit anti-p65 (Santa Cruz Biotechnology, sc-372), rabbit anti-p65 (Cell signaling, 8242), rabbit anti- β-catenin (gift from Dr. Peter Klein).

Goblet cell secreted mucins were identified by sequentially incubating deparaffinized sections in pH 2.5 alcian blue (1 hour), periodic acid (7 minutes) and Schiff's reagent (10 minutes). After the staining, acidic mucins are stained “blue” and neutral mucins are stained red.

Fluorescence in situ Hybridization (FISH) was performed to detect eubacteria infiltration in the colon. Paraffin sections (10 μm) were dewaxed and incubated with the commercially synthesized universal eubacterial probe EUB 338 (5′-GCTGCCTCCCGTAGGAGT-3′) conjugated with Alexa Fluor 488 as described [47]. A complimentary probe (5′-ACTCCTACGGGAGGCAGC-3′)) conjugated with Alexa Fluor 488 was used as a negative control. Sections were counterstained with DAPI afterwards.

Images were taken from a Compound microscope (Leica) with digital camera or a Nikon A1R confocal microscope and processed using Adobe Photoshop.