Lsm 780 laser confocal microscopy

Six rats of each group were selected at random for immunofluorescence. After collection, tissues of were fixed in 4% paraformaldehyde and embedded in paraffin. Blocks were sectioned (5 μm), mounted on slides, then deparaffinized and rehydrated by successive incubations in xylene, 100% ethanol, 95% ethanol, 80% ethanol, 70% ethanol, and phosphate buffer saline (PBS). Custom anti-occludin antibodies (Abcam, Cambridge, MA, USA) were applied overnight at 4°C, followed by an Alexa 488–conjugated goat anti-rabbit IgG antibody for 60 min (Abcam, Cambridge, MA, USA). The slides were washed extensively and stained with DAPI (4’6’-diamidino-2-phenylindole) (Abcam, Cambridge, MA, USA). Images were obtained (Zeiss LSM 780 laser confocal microscopy) at excitation wavelengths of 390 nm and 500 nm; emission was detected at 440 and 580 nm. All the stainings were performed in duplicate in non-serial distant sections, and analyzed in a double-blind manner by two different experienced investigators.

The resected mice colon tissues were washed with cold PBS followed by immediate fixation in 4% PFA then stored at 4 °C overnight. For histopathological analysis, paraffin-embedded tissue sample were sectioned (4 µm) and stained with hematoxylin and eosin (H and E). All images of sections were obtained using a BZ-X710 All-in-one inverted fluorescence microscope (Keyence, Osaka, Japan) with 10× and 20× objective lenses. The images were analyzed using BZ-X analyzer software (Keyence) and ImageJ software (NIH, Bethesda, MD, USA). For the immunohistology of F4/80 positive cells in colon tissues, frozen OCT-embedded tissue samples were sectioned (8 µm) at −20 °C then post-fixation with 4% PFA for 10 min. Permeabilization was performed with 0.5% triton x-100 in PBS with 15 min. The sections were blocked by 10% goat serum (Nichirei Bioscience, Tokyo, Japan) for 30 min. Immunostaining of F4/80 was performed with APC conjugated F4/80 antibody (123115, BioLegend, San Diego, CA, USA) at 4 °C, overnight. Sections were mounted with Prolong gold antifade mountant with DAPI (Thermo Scientific, Waltham, MA, USA). The images were captured with LSM-780 confocal laser microscopy (Carl Zeiss, Jena, Germany). The excitation wavelength was set with 633 nm laser for APC detection and 405 nm laser for DAPI. The images were analyzed using Zen software (Carl Zeiss).