Nis elements br 3.2 imaging software

Samples were fixed in 2% PFA overnight and soaked in phenol for 6 days before paraffin embedding and cross-sectioning (as modified from Li et al. 2015) [23 (link)]. Prior to paraffin embedding, insects were re-fixed with formaldehyde then dehydrated in an ethanol gradient series. Samples were stored until sectioning. Transverse sections were taken using a Microm HM 325 rotary microtome (Walldorf, Germany) and slides hand-stained in Harris hematoxylin and eosin-phloxine. Slides were imaged using a Nikon Eclipse E600 compound microscope (Nikon Instruments, Melville, NewYork) and Nikon Digital Sight DS-Ri1 high-resolution microscope camera through Nikon NIS-Elements BR 3.2 imaging software.

Monolayers of DPSCs were cultured with DMEM containing 10 % FBS for 48 h in four-well covered glass chamber slides. After two washes with phosphate-buffered saline (PBS) containing 1 % bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA), cell surface Fc receptors were blocked with immunoglobulin G (IgG) (Santa Cruz Biotechnology Inc., Dallas, TX, USA) on ice for 15 min. The cells were then stained for 30 min at 37 °C with a 1:100 dilution of a fluorescein isothiocyanate (FITC)-conjugated anti-CD44 monoclonal antibody (BD Biosciences, Franklin Lakes, NJ, USA) or an isotype-matched FITC-conjugated IgG control antibody (BD Biosciences). After washing, the cells were analyzed using an ECLIPSE TS100-F microscope equipped with an Intensilight C-HGFIE illumination system (Nikon Co., Ltd., Tochigi, Japan). Digital images were processed with NIS Elements BR3.2 imaging software (Nikon Co., Ltd.) and Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA, USA).

FISH in the distal colonic section was performed as described previously38 (link), except for using 10 μg of an Alexa Fluor 488-conjugated bacterial rRNA EUB338 probe for hybridization. The epithelial region in contact with the bacteria was evaluated on a FISH-stained section, and the proportion of epithelial region in contact with bacteria was determined from a whole intestinal section in a blind fashion using NIS-Elements BR 3.2 imaging software (Nikon). For co-immunostaining, the FISH-stained slides were blocked and incubated with anti-Muc2 antibody (sc-15334, Santa Cruz Biotech.) overnight at 4 °C. The slides were further incubated with goat anti-rabbit Alexa 568 (Life Technologies), counterstained with DAPI solution (0.5 μg/ml in PBS) and visualized with a confocal microscope.