IHC and IF analyses were performed as described previously (49 (link)). Primary antibodies for IHC and IF were as followed: anti-MUC2 (GB14110; 1:500; Servicebio); anti-MPO (GB12224; 1:200; Servicebio); anti-Occludin (GB111401">GB111401 ; 1:200; Servicebio). Data were analyzed by Image J.
Gb111401
Manufactured by Wuhan Servicebio Technology
Sourced in China
GB111401 is a laboratory instrument designed for cell culture applications. It provides a controlled environment for the growth and maintenance of cells. The core function of this product is to regulate temperature, humidity, and gas composition to support optimal cell culture conditions.
Automatically generated - may contain errors
Lab products found in correlation
Gb11032, by Wuhan Servicebio Technology (5 mentions)
Gb111981, by Wuhan Servicebio Technology (3 mentions)
Gb12224, by Wuhan Servicebio Technology (2 mentions)
Anti muc2, by Wuhan Servicebio Technology (2 mentions)
Gb14110, by Wuhan Servicebio Technology (2 mentions)
Anti mpo, by Wuhan Servicebio Technology (2 mentions)
Anti occludin, by Wuhan Servicebio Technology (2 mentions)
Gb11195, by Wuhan Servicebio Technology (2 mentions)
Gb23303, by Wuhan Servicebio Technology (2 mentions)
Gb111402, by Wuhan Servicebio Technology (2 mentions)
Dy07 microscope, by Olympus (1 mentions)
Bl003a, by Biosharp (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Gb12082, by Wuhan Servicebio Technology (1 mentions)
Gb12192, by Wuhan Servicebio Technology (1 mentions)
Vectra polaris imaging system, by Akoya Biosciences (1 mentions)
Gb11181, by Wuhan Servicebio Technology (1 mentions)
G1211, by Wuhan Servicebio Technology (1 mentions)
G1004, by Wuhan Servicebio Technology (1 mentions)
Eclipse c1 fluorescence microscope, by Nikon (1 mentions)
13 protocols using gb111401
1
Immunohistochemical and Immunofluorescence Analysis
2
Immunohistochemical and Immunofluorescence Analysis
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IHC and IF analyses were performed as described previously (49 (link)). Primary antibodies for IHC and IF were as followed: anti-MUC2 (GB14110; 1:500; Servicebio); anti-MPO (GB12224; 1:200; Servicebio); anti-Occludin (GB111401">GB111401 ; 1:200; Servicebio). Data were analyzed by Image J.
3
Immunohistochemical Analysis of Tight Junctions
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The colons were fixed in 4% paraformaldehyde for four hours and then transferred to 70% ethanol for preservation. Subsequently, the samples were sliced into four-μm-thick sections and embedded in paraffin. These colon sections were subjected to overnight incubation at 4°C with specific primary antibodies: Recombinant anti-Claudin-1 antibody (GB15032, Servicebio, China) at a dilution of 1:500 (Mouse mAb), anti-Occludin Rabbit pAb (GB111401, Servicebio) at a dilution of 1:500, or anti-ZO-1 tight junction protein Rabbit pAb (GB11195, Servicebio) at a dilution of 1:500. Following this, the sections were treated with a goat anti-rabbit secondary antibody (Beijing Zhong Shan Golden Bridge Biotechnology Co., Ltd., China) for one hour at room temperature. The sections were examined using an Olympus DY07 microscope (Olympus, Japan) and high-resolution images were captured using a camera at a magnification of 400.
4
Intestinal Tight Junction Protein Analysis
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The protein expression levels of ZO-1, claudin 1, and occludin in the intestinal tissues of mice were assessed via Western blotting. The total protein in each intestinal tissue was evaluated. Then, the samples were added with 6 × loading buffer and were boiled for 6 min. Next, 12% and 10% separating gel and 5% concentrated gel were prepared, and 50 µg of protein was loaded into each well. The total proteins were separated via polyacrylamide gel electrophoresis, and it was transferred to a PVDF film using the wet method. Seal milk powder with 5% skimmed for 2 hours. After dilution with ZO-1 (BIOSS, bs-1329R), claudin 1 (Servicebio, GB112543), and occludin (Servicebio, GB111401) based on the manufacturer’s instructions, add them to the PVDF film to ensure that the film is completely covered. The samples were incubated overnight in a shaker at a temperature of 4 °C. After rinsing with Tris buffered saline + Tween 20 on the second day, the dilution was added (Biosharp, BL003A), incubated at room temperature for 2 h, and ECL reagent was added after rinsing was completed. Next, the gray value on the strip was read using the Image J software.
5
Immunohistochemical Analysis of PPARα and Occludin
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The hepatic PPARα and intestinal Occludin expression were evaluated using paraffin-embedded liver and ileum tissues, respectively. After deparaffinization, the slides were heated in an autoclave with sodium citrate for antigen repairing, followed by 1% hydrogen peroxide to abolish endogenous peroxidase activity, and blocked with 2% goat serum. Slides were then incubated with primary antibodies including PPARα (Servicebio, GB11163, 1 : 20) and Occludin (Servicebio, GB111401, 1 : 500) at 4°C overnight. HRP-conjugated secondary antibodies (1 : 200) were incubated for 50 min at room temperature. After PBS washing, peroxidase substrate DAB (Dako, K5007) was used for color development.
6
Immunohistochemical Analysis of Colon Tissue
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The sections of colon tissue were blocked by 3% H2O2 and goat serum; then, the samples were incubated with the following primary antibodies at 4°C overnight: ZO-1 (1 : 200, GB111981, Servicebio), occludin (1 : 600, GB111401, Servicebio), claudin-1 (1 : 400, GB11032, Servicebio), E-cadherin (1 : 500, GB12082, Servicebio), and vimentin (1 : 1000, GB12192, Servicebio). Then, sections were washed with PBS and incubated with the appropriate secondary antibody (Dako Real Envision/HRP, Rabbit/Mouse, K5007) at room temperature for 1 h. After that, 3,3′-diaminobenzidine with peroxidase substrate (Dako Real DAB Chromogen, K5007) were used for chromogenic reaction. Counter staining was performed with hematoxylin.
7
Immunohistochemical Analysis of Brain and Intestine
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For immunohistochemistry, tissues (brains and intestines) were sectioned at 30 μm thickness and treated with methanolic H2O2 for 30 min. The sections were incubated with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 15 min and blocked with 4% normal serum in PBS for 15 min before incubating with the primary antibody. The sections were incubated overnight with TH (1:2,000, Servicebio, GB11181), ZO-1 (1:300, Servicebio, GB111981), occludin (1:500, Servicebio, GB111401), or claudin (1:300, Servicebio, GB11032). The sections were washed three times in PBS (pH 7.4, and incubated with secondary antibodies (1:200, Servicebio, GB23303) for 50 min at room temperature. After the slices were slightly spin-dried 3 times, 3,3′-diaminobenzidine tetrahydrochloride (DAB) condensed chromogen (Servicebio, G1211) was utilized to visualize. After immunostaining, sections were counterstained with hematoxylin (Servicebio, G1004). Sections were photographed using the Vectra Polaris Imaging System (Akoya).
8
Immunofluorescence Analysis of Intestinal Tight Junctions
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The 5-µm-thick paraffin-embedded jejunal and ileal tissue sections were used for immunofluorescence. Heat-induced antigen retrieval was performed by autoclaving the sections for 10 minutes at 121°C in 10mM sodium citrate buffer (pH 6.0). The sections were blocked with 8% skim milk in TBST at 37°C for 40 minutes, and then immunostained using primary antibodies against ZO-1 (1:200, GB11195, rabbit; Servicebio), occludin (1:200, GB111401, rabbit; Servicebio), claudin-1 (1:200, GB11032, rabbit; Servicebio) at 4°C overnight. The sections were washed and incubated with secondary fluorescent antibodies at 37°C for 60 minutes. The secondary antibodies was CY3 goat anti-rabbit IgG (1:300; GB21303; Servicebio). Sections were mounted with Nikon DS-U3 with DAPI (G1012, Servicebio). Images were captured with an Nikon Eclipse C1 fluorescence microscope (Nikon, Tokyo, Japan).
9
Histological Analysis of Intestinal Tight Junctions
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The paraffin-embedded tissues were sectioned into 3-μm-thick slides. According to standard histological protocols, hematoxylin and eosin (HE) staining was conducted. For immunohistochemistry (IHC), the following primary antibodies were used to incubate the slides, respectively, after blocking: bacterial lipopolysaccharide (LPS; HycultBiotech #HM6011, Netherlands), lipoteichoic acid (LTA; Santa Cruz, #sc-57752, United States), occludin (Servicebio #GB111401, China), and zonula occludens 1 (ZO-1; Servicebio #GB111981, China). IHC quantification involved the calculation of positive areas (occludin and ZO-1) using Aipathwell software (Version 2.0, Servicebio, China).
10
Immunofluorescence Analysis of Tight Junction Proteins
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Mice were transcardially perfused with 0.1 M phosphate buffer (PB) followed by 4% paraformaldehyde (PFA) under deep anesthesia. The distal colons were removed, postfixed in 4% PFA and embedded with OCT. The sections (10 μm) were blocked with 10% normal goat serum in 0.05 M PBS and were incubated with one of the following primary antibodies at 4°C overnight: 1) rabbit anti-occludin (1:1500; GB111401; Servicebio, Shanghai, China); 2) rabbit anti-claudin-1 (1:500; GB11032; Servicebio); and 3) rabbit anti-ZO-1 (1:1500; GB111402; Servicebio). The following day, sections were incubated with goat anti-rabbit Alexa Fluor 488 secondary antibody (1:1000; Molecular Probes-Invitrogen, Eugene, USA) at room temperature for 1 h. The sections were viewed under a fluorescence microscope (Leica DM2500; Leica, Wetzlar, Germany), and digital images were captured using Leica Application Suite version 4.3 (Leica). Integrated density was measured to evaluate fluorescent signals using ImageJ (
http://rsb.info.nih.gov/ij/ ).
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