Eclipse ti u microscope

Aliquots (100 μl) of Matrigel® Growth Factor Reduced (GFR) Basement (Catalog # 354230; Corning, USA) were plated into individual wells of 96-well tissue culture plates (Becton Dickinson) and allowed to polymerize overnight at 37 °C in a cell culture incubator. In the tube formation experiments, three treatment groups were used. Group one consisted of HUVECs cultured alone. Group two consisted of HUVECs cultured with 100 mM glucose. Group three consisted of HUVECs cultured with 100 mM glucose and 25% CMpMSC. Group four consisted of HUVECs cultured with 100 mM glucose and pMSCs. Varying pMSC:HUVEC ratios (1:30, 1:6, and 1:4) were used.
HUVECs were seeded at a density of 3 × 10⁴ cells per well in a complete endothelial cell growth culture medium on the polymerized Matrigel. Following 14 h, the tube network formed was observed under an inverted Nikon ECLIPSE Ti U microscope (Nikon, Japan). Photomicrographs were recorded using a Nikon DS-Qi1 camera and data were analysed with the software (Nikon, Japan). Experiments were carried out in triplicate and repeated as already described.

After CLP surgery 48 h later, mice were sacrificed and the colonic and ileac segments 2 to 3 cm in length were excised, washed in phosphate buffered saline (PBS), fixed in 4% formaldehyde, embedded in paraffin, and sectioned (5-μm thick). A portion of the paraffin sections were stained with hematoxylin and eosin (H&E) and the rest was used for immunofluorescence using procedures detailed previously (32 (link)). Briefly, antigens were unmasked by boiling under pressure in sodium citrate buffer. Tissue sections were cooled to room temperature (about 20°C) naturally and then washed with PBS three times (5 min each wash). The sections were incubated with a blocking buffer (5% donkey serum in PBS) for 60 min against the species of the secondary antibody. Primary antibodies (Mucin 2 H-300, rabbit polycional lgG, Santa Cruz, USA) were incubated at overnight 4°C and then the sections were washed three times with PBS. Alexa Fluor® 488 and 594, donkey anti-rabbit lgG (H+L; Life, USA) were diluted 1:1000 and incubated for 1 h at RT and washed three times in PBS. Nuclei were counterstained using DAPI. The tissue sections were then photographed with Nikon Eclipse Ti-U microscope and Nikon Intensilight C-HGFI (Japan).

Histologic section of the liver in mice was treated with hematoxylin-eosin (H&E) staining or mason staining as we described before [3 (link), 25 (link)]. Then, the sections were observed under a Nikon Eclipse Ti-U microscope (Nikon, Tokyo, Japan).