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5 protocols using villin

1

Antibody Immunofluorescence Protocol

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The primary antibodies, AANAT (17990–1-AP) was purchased from Proteintech, and MyD88 (ab28763) and Villin (ab201989) were purchased from Abcam. Immunofluorescence was performed according to our previous protocols.61 (link)
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2

Protein Expression Analysis by Western Blotting

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Protein samples were prepared via homogenization of cells in lysis buffer (10 mmol/L Tris-HCl, pH 8.0; 140 mmol/L NaCl; 5 mmol/L EDTA; 0.25 g/L NaN3; 10 g/L Triton X-100; 10 g/L deoxycholate; 1 g/L SDS; 0.5 mmol/L PMSF; 1 g/L leupeptin; and 1 g/L aprotinin). The protein concentration was determined using the Coomassie brilliant blue method. Proteins were separated via SDS-PAGE, transferred to PVDF membranes (Millipore, Temecula, CA, USA), blocked with milk/BSA, and then probed with specific antibodies against KLF5 (1:1000), CDX2 (1:800), MUC2 (1:1000), villin (1:1000) (all from Abcam, Cambridge, MA, USA) and GAPDH (1:2000, Kangchen, Shanghai, China). After washing, the blots were incubated in a horseradish peroxidase-conjugated goat anti-mouse secondary antibody, then visualized using an enhanced chemiluminescence reagent (Pierce Biotechnology, Rockford, IL, USA), and subsequently exposed to autoradiographic film. All experiments were reproduced at least three times. The western blotting bands were analyzed using ImageJ software (Bio-Arts, Co., Ltd., Fukuoka, Japan).
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3

Immunofluorescence Analysis of Colon Tissues

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Control and C. rodentium treated mice samples of the proximal colon were frozen and cut into small sections (~5 μm). Then, at room temperature they were placed in 4% paraformaldehyde in PBS for a duration of 10 min. Prior to incubation, fixed sections were washed in PBS, 0.3% Nonidet P-40 for 5 min was used for permeabilization, followed by 5% normal goat serum (NGS) for 2h for blocking of the sections. Tissues were then incubated at room temperature with the primary antibodies, P-gp (Santacruz) and villin (Abcam) (1:100) in PBS composed of 1% NGS for a duration of 2h. After the primary washings, sections were incubated for a duration of 1h with the secondary antibodies, Alexa Fluor 594 and 647 conjugated goat anti-rabbit IgGs (1:100, Invitrogen) in 1% NGS. After the secondary washes of the sections, slow-fade diamond antifade with DAPI reagent (Invitrogen) was used to position the sections under coverslips. Analysis of the sections was performed using an Olympus BX fluorescent microscope equipped with 20X objective for imaging.
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4

Western Blot Analysis of Collagen and Villin

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Harvested IECs were lysed in triton lysis buffer (137 mM NaCl, 20 mM Tris base at pH 7.4, 10% glycerol and 1% Triton X-100) supplemented with a protease and phosphatase inhibitor mixture (Roche) for 20min on ice, and centrifuged at 15,000 rpm for 10 min at 4°C. 10 μg of denatured proteins were loaded onto a Mini-PROTEAN Precast Gel (Bio-Rad) and transferred onto a PVDF membrane (Thermo Fisher Scientific).The membranes were blocked by 5% skim milk in TBST (Tris-buffered saline /0.1% Tween 20, Bio-Rad) and then incubated with antibodies to collagen a1 (IV) (Origene) and Villin (Abcam). Immunoreactive bands were detected by chemiluminescence (ECL solution, Santa Cruz).
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5

Histological Analysis of Intestinal Tissues

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Intestinal tissues of mice and minipigs were fixed with 10% neutral buffered formalin solution, embedded in paraffin wax, and sectioned transversely at a thickness of 4 µm. The slides were stained with H&E and Congo red. Histological scores were quantified in the H&E-stained slides and assessed by the degree of the epithelial maintenance, crypt damage, vascular dysfunction, and inflammation with infiltration of inflammatory cells. To investigate radiation-induced goblet cell damage, the slides were stained with PAS. For immunohistochemical analysis, the slides were subjected to antigen retrieval for 20 min and then treated with 0.3% hydrogen peroxide in methyl alcohol for 20 min to block endogenous peroxidase activity. After washing with PBS, the slides were blocked with 10% normal goat serum (Vector ABC Elite kit; Vector Laboratories, Burlingame, CA, USA) and incubated with primary antibodies, such as anti-Cldn3 (Invitrogen, Carlsbad, CA, USA), villin (Abcam, Cambridge, UK), Mpo (Abcam), ki-67 (Acris), Olfm4 (Invitrogen), and CD68 (Abcam) antibodies. Additionally, the slides were incubated with horseradish peroxidase-conjugated secondary antibody (Dako, Carpinteria, CA, USA) for 60 min. The peroxidase reaction was developed using diaminobenzidine substrate (Dako) prepared according to the manufacturer’s instructions, and the slides were counterstained with hematoxylin.
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