Occludin

After fish were euthanized by decapitation, the intestine was immediately dissected, fixed in a 10% paraformaldehyde solution (12 h), washed in PBS buffer (0.1 M, pH 7.2) and dehydrated with a 70% ethanol gradient and embedded in paraffin (Paraplast/McCormick) The samples that were embedded in paraffin were sectioned to a thickness of 5 μm. The sections were stained with haematoxylin for 5 min, washed with PBS and then stained with eosin at room temperature (3 sections per fish); In order to observe intensity of goblet cells, the sections were stained with periodic-acid schiff (PAS) (3 sections per fish).
For immunohistochemistry, the samples that were embedded in paraffin were sectioned to a thickness of 5 μm. After deparaffinization and hydration, the sections were pre-treated with 3% H₂O₂ in methanol at room temperature for 10 min and then heated in 10 mmol/L citrate buffer (pH 6.0). After several rinses in PBS, the sections were blocked with 10% goat serum at room temperature for 20 mins, and then incubated overnight at 4°C primary antibody (MUC2, No. ab272692, Abcam; GPR 78, No.11587-1-AP, Proteintech; occludin, No. 27260, Proteintech). After rinsing with PBS several times, the sections were incubated with a biotinylated secondary antibody at 37°C for 30 min. After rinsing several times in PBS, immunodetection was conducted.

The fruiting body of the mushroom Dictyophora indusiata was obtained from Anhui Joy Lok Food Co., Ltd., Ningde, Fujian Province, China. The broad-spectrum antibiotics clindamycin hydrochloride and metronidazole were from Tian Fang Pharmaceutical Co., Ltd. (Zhumadian, China) and Changchun Wan de Pharmaceutical Co., Ltd. (Changchun, China) respectively. The DNA extraction kit (QIAamp DNA Stool Mini Kit) and gel purification kit (Agencourt AMPure XP 60 mL Kit) were from Qiagen (Hilden, Germany) and Beckman Coulter (Brea, CA, USA), respectively. The primary antibodies (β-actin, claudin-1, occludin, and zonula occludens-1 (ZO-1)), secondary antibodies, and the Radioimmunoprecipitation assay (RIPA) buffer were from Proteintech (Wuhan, China). The Mucin-2 (MUC2) primary antibody was from Boster Biological Technology, Wuhan, China. The horseradish peroxidase-conjugated secondary antibody and DAB substrate chromogen system were from ZSGB-BIO (Beijing, China). The bicinchoninic acid (BCA) protein assay kit was from Pierce Rockford, IL, USA. The Limulus amebocyte lysate (LAL) assay kit was obtained from Chinese horseshoe crab reagent manufactory Co., Ltd. (Xiamen, China). The ELISA kits were purchased from Shang Hai Lengton Bioscience Co, Ltd., Shanghai, China. All the other chemicals used in this study were of analytical grade and purchased from the standard commercial sources.

Distal colon segments in the same defined region were collected carefully and were transferred immediately to 10% neutral-buffered formalin. After paraffin embedding, colon fragments were generated into 5 μm cross-sections. The sections were stained with hematoxylin and eosin (H&E) and then scanned by a NanoZoomer Digital Pathology system (Hamamatsu Photonics, KK, Japan) for further histopathological analysis. At least three slides were randomly selected and observed by a blinded pathologist. The histological activity index (HAI) is an established scoring system based on inflammatory cell infiltration (1–3), both in terms of severity and extent, and intestinal architecture (1–3), which includes epithelial changes and mucosal architecture, as previously described (Erben et al., 2014 (link)).
The colon sections were stained with the intestinal barrier markers ZO-1 and Occludin (both Proteintech, Rosemont, IL, United States) with a previously described standard immunofluorescence analysis process (Chung et al., 2014 (link)). Images were captured and visualized using a Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).

After routine dewaxing and antigen reparation, the sections were sealed with 3% bovine serum albumin, and incubated with Occludin (Cat No.66378-1-Ig, Proteintech) or Claudin-1 (Cat No.13050-1-AP, Proteintech) or ZO-1 (Cat No.21773-1-AP, Proteintech), or CD-31 (Cat No. NB100-1642, Novusbio) antibody (1:200) at 4 °C overnights. Then sections were washed with PBS three times and incubated with secondary antibody (Alexa Fluor 488, cy3, Proteintech, 1:200) for 2 h at 37 °C. Photographs were taken using an immunofluorescent microscope (Nikon, Japan) and analyzed relative %area positive expression by software ImageJ.

The fruiting body of the mushroom D. indusiata was obtained from Anhui Joy Lok Food Co., Ltd., Ningde, Fujian Province, China. The DNA extraction kit (QIAamp DNA Stool Mini Kit) and gel purification kit (Agencourt AMPure XP 60 mL Kit) were from Qiagen (Hilden, Germany) and Beckman Coulter (Brea, CA, USA), respectively. The primary antibodies β-actin, claudin-1, occludin, zonula occludens-1 (ZO-1), secondary antibodies, and the Radioimmunoprecipitation assay (RIPA) buffer were from Proteintech (Wuhan, China). The horseradish peroxidase-conjugated secondary antibody was from ZSGB-BIO (Beijing, China). The bicinchoninic acid (BCA) protein assay kit was from Pierce Rockford, IL, USA. Polyvinylidene difluoride (PVDF) membranes were from Immobilon TM-P, Millipore, Massachusetts, USA, and WesternBright™ ECL substrate was from Advansta, Inc., Menlo Park, CA, USA. The ELISA kits were purchased from Shang Hai Lengton Bioscience Co, Ltd., Shanghai, China. All the other chemicals used in this study were of analytical grade and purchased from standard commercial sources.