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Up. # p 0.05, ## p 0.01, ### p 0.001 relative to control group; p 0.01, p 0.001, relative
Up. # p 0.05, ## p 0.01, ### p 0.001 relative to control group; p 0.01, p 0.001, relative to DSS group. (D) Photos of colon samples displaying the colon length. (E) Colon length. (F) Colon weight/length ratio. (G) Histological scores on the H E-stained sections. (H) H E-stained colonic sections. Scale bar = 100 . Data are imply SEM. n = 6 per group. p 0.05, p 0.01, p 0.001 and p 0.0001.Nutrients 2021, 13,four of2.4. Quantitative Haloxyfop supplier real-time PCR Total RNA was extracted from the colonic tissue applying Trizol (Invitrogen, Carlsbad, CA, USA). cDNA was obtained using the GoScript reverse transcription system (Promega, Madison, WI, USA). A real-time PCR technique (ABI 7500) was used to execute qPCR. The sequences on the primers applied in this study are listed in Table S3. The relative expression on the target genes was calculated employing GAPDH because the reference gene. 2.5. Western Blotting Colon tissues have been lysed making use of a radioimmunoprecipitation assay (RIPA). The protein concentration was detected working with the BCA protein assay kit (Life Technologies, Eugene, OR, USA). Thirty milligrams on the proteins were separated by electrophoresis on a ten SDS polyacrylamide gel then transferred to PVDF membranes. The samples were incubated with key antibodies against phospho-ERK1/2 (Cell Cephapirin Benzathine Bacterial Signaling Technologies, Boston, MA, USA), anti-total ERK1/2 (Cell Signaling Technology), phospho-JNK (Cell Signaling Technology), anti-total JNK (Cell Signaling Technologies), phospho-p38 (Cell Signaling Technology), anti-total p38 (Cell Signaling Technologies), phospho-NF-B (Cell Signaling Technologies), anti-total NF-B (Cell Signaling Technology), and HSP90 (Cell Signaling Technologies). Proteins had been detected using the HRP-conjugated secondary antibody along with the chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). 2.six. Immunofluorescent Staining Colon tissues have been permeabilized utilizing Tris-buffered saline (TBS) containing 0.three Triton X-100 and incubated at room temperature for 30 min. The tissues have been then boiled in Tris EDTA (pH 9.0) for 20 min, blocked with 5 bovine serum albumin in TBS (pH 7.4) for 1 h, then incubated overnight with all the primary antibody ZO-1 (Abcam, Cambridge, MA, USA), Occludin (Santa Cruz, Dallas, TX, USA), and Muc2 (Santa Cruz, Dallas, TX, USA) and incubated at four C prior to the AlexaFluor-488 secondary antibody (Invitrogen, Carlsbad, CA, USA) was utilized. two.7. Hematoxylin and Eosin (H E Staining) Colon tissues have been fixed in 4 paraformaldehyde and embedded in paraffin. The paraffin sections had been cut into four -thick sections that have been stained with H E and observed applying an optical microscope. The histopathologic score was obtained in accordance with the following evaluation criteria: Crypt damage (0 scale), the severity of inflammation (0 scale), and the depth of injury (0 scale) [18]. 2.eight. Alcian Blue Staining Fresh colon tissue sections have been fixed in ten buffered formalin. Paraffin was used to embed the tissues and also the tissues had been stained with Alcian-Blue/Nuclear-Fast-Red for 20 min following dewaxing the tissue section. Goblet cells had been viewed beneath a light microscope (Leica DM500, Chicago, IL, USA). The quantification of goblet cells determined by Alcian bluestained sections was performed using Image J computer software. Six crypts of every single section have been randomly selected to evaluate the goblet cell number of crypts. 2.9. Transmission Electron Microscopy Colonic tissues were fixed with two.five glutaraldehyde straight away soon after the mice were sacrificed. The excess fixativ.