Fv10 asw viewer

FISH assays were performed using a RiboTM lncRNA FISH probe Mix Kit (cat. no. c10910; Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. Oligonucleotide modified Cy-3-labeled probes for VIM-AS1 (5′-TAG GAC TTC CTA GTA CTT CTG A-3), GAPDH and U6 were designed and synthesized by Genecreate. C4-2 cells were seeded on 20-mm confocal dishes (Corning, Inc). After overnight incubation, C4-2 cells were fixed with 4% paraformaldehyde for 20 min at 4°C and permeabilized using Triton X-100 for 90 sec at 4°C. Next, 250 µl prehybridization solution with 1% blocking solution (Guangzhou RiboBio Co., Ltd.) was added to C4-2 cells and cells were incubated at 42°C for 1 h. Subsequently, C4-2 cells were incubated with 100 µl hybridization buffer (Guangzhou RiboBio Co., Ltd.) supplemented with 1% blocking solution and 2.5 µl 20 µM 22-nucleotide CY-3-labeled-VIM-AS1, CY-3-labeled-GAPDH or CY-3-labeled-U6 FISH probe at 37°C overnight in a dark moist chamber. The following day, cells were washed three times in 2X sodium citrate buffer for 5 min at 42°C and stained with DAPI at 4°C for 10 min. Images were acquired using a laser scanning confocal microscope (FV1000; Olympus Corporation) and corresponding software (FV10-ASW Viewer; version 4.2; Olympus Corporation) at a magnification of ×400.

Labeled tissues were examined with a laser scanning confocal microscope using a 20× (NA 0.95) XLUMPlanFl objective (Olympus Japan) in Fluoview (Olympus Japan) using the optimal confocal aperture to give a resolution of 0.994 × 0.994 × 1.13 μm (X × Y × Z). Stacks of confocal images across the full thickness of the muscularis propria were collected from three nondiabetic Csf1^op/op mice (20–22 weeks old), three diabetic Csf1^op/op mice (20–22 weeks old), three nondiabetic Csf1^+/+ mice (20–22 weeks old), and three diabetic Csf1^+/+ with normal GE and 3 diabetic Csf1^+/+ with delayed GE mice (2, 3, and 4.6 weeks after onset of diabetes). Six images from the gastric body and three from the antrum were taken for each animal. For quantification of the labeling, all of the confocal image stacks were flattened into projections using the FV10-ASW Viewer (Olympus). The flattened images were renumbered in random order and then assessed by scoring the integrity of the ICC on a 10-cm visual analogue scale for network density. Scoring was done in random order by two independent investigators blind to the image and tissue source.