Protein samples were obtained from cells using RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail. Twenty micrograms of protein was separated using 10% SDS–PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with blocking buffer at room temperature for 15 min, PVDF membranes were incubated with primary antibodies, including anti-PD-L1 (1:2 000, Proteintech, China), anti-Claudin-1 (1:2 000, Proteintech, China) and anti-GAPDH (1:4 000, Proteintech, China), at 4 °C overnight. Then, they were incubated with the corresponding secondary antibody for 1–2 h at room temperature. The protein bands were visualized using the BeyoECL Moon detection system. For quantitative analysis of protein expression, ImageJ software was applied to measure the optical densities of the blot bands.
Anti claudin 1
Manufactured by Proteintech
Sourced in China
Anti-Claudin-1 is a primary antibody that detects the Claudin-1 protein. Claudin-1 is a tight junction protein that plays a role in epithelial cell-cell adhesion. This antibody can be used to detect Claudin-1 expression in various samples using techniques such as Western blotting, immunohistochemistry, or immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti zo 1, by Proteintech (3 mentions)
Anti occludin, by Proteintech (2 mentions)
Anti pd l1, by Proteintech (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ecl western blotting substrate, by Bio-Rad (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Pierce bca protein assay kit, by Beyotime (1 mentions)
Image j analysis system, by Bio-Rad (1 mentions)
Anti occludin 1, by Proteintech (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Proteintech (1 mentions)
5 protocols using anti claudin 1
1
Western Blot Analysis of PD-L1, Claudin-1, and GAPDH
2
Intestinal Protein Expression Analysis
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Intestine tissue (n = 4 pups/group) soaked in RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitor (Beyotime, China) was homogenized using an electric homogenizer and centrifuged to obtain the supernatant. Protein concentrations were measured using a Pierce BCA Protein Assay Kit (Beyotime, China). The protein supernatant was mixed with sodium dodecyl sulfate sample buffer (Beyotime, China) at a ratio of 4:1 and denatured at 100 ℃ for 10 min. Protein samples were separated in 10% polyacrylamide gels and transferred to 0.45 μm PVDF membranes, and measured using anti-TLR4, anti-NF-κB P65 (Servicebio, China), anti-ZO-1, anti-Occludin, anti-Claudin-1 (Proteintech, China) and β-Actin (ZENBIO Biotechnology, China) at 4 ℃ overnight. Signals were detected using chemiluminescence (ECL Western Blotting Substrate, Bio-Rad). The relative intensity of target bands was quantified using the Image J analysis system (Bio-Rad).
3
Intestinal Tight Junction Protein Analysis
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In brief, intestinal mucosal samples were homogenized in 1 mL lysis buffer and centrifuged at 12,000×g at 4°C for 30 min for supernatants collection, and then the protein concentrations were measured using phenol reagent method. An equal amount of proteins samples (20 to 40 μg) was separated on a 10% or 12% reducing polyacrylamide gel and transferred onto polyvinylidene difluoride membranes, and incubated with primary antibodies. Immunoblots were blocked with 3% bull serum albumin (BSA) in Tris-buffered saline for 70 min at room temperature and incubated overnight at 4°C with the specific primary antibodies in Tris-buffered saline and 0.05% Tween 20 containing 1% BSA. The specific primary antibody included rabbit anti-claudin-1 (1:1,000; Proteintech, Chicago, IL, USA) and anti-occludin-1 (1:1,000; Proteintech, USA). Blots were washed with the same buffer and then incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000; Proteintech, USA) for 120 min at room temperature. Membranes were washed in the same buffer and antigen-antibody complexes visualized by using an ECL kit (Pierce, Waltham, MA, USA). The density of the bands was quantified using the Image J 1.46r analysis software.
4
Histological Analysis of Liver, Kidney, and Colon
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Liver, kidney and colon tissues were fixed in 4% paraformaldehyde, routinely dehydrated, embedded and cut into sections of 4 μm thick. The tissue sections were stained with hematoxylin and eosin (H&E), oil red O, periodic acid Schiff (PAS) and Masson staining by kits (Servicebio, Wuhan, China) following manufacturer’s instructions. The percentage of mesangial hyperplasia was calculated as the ratio of the pink mesangial area and the total glomerular area in each glomerulus. The quantitative statistical method of collagen area from Masson staining: Collagen area fraction (%) = (MASSON staining collagen positive area/total area) * 100%”. Each group randomly selected four fields of vision, blinded by two researchers (magnification: 400×).
The colon tissue sections were routinely deparaffinized and antigen retrieval was performed with EDTA and blocked with 3%BSA. Primary antibodies including anti‐ZO‐1 (catalog no. 66452‐1; 1:100; ProteinTech Group) and anti-Claudin-1 (catalog no. 13050-1; 1:100; ProteinTech Group) primary antibody were applied for incubation at 4°C overnight followed by incubation of the corresponding secondary antibody (1:200) for an hour at room temperature, and then mounted with a medium containing DAPI for capturing pictures.
The colon tissue sections were routinely deparaffinized and antigen retrieval was performed with EDTA and blocked with 3%BSA. Primary antibodies including anti‐ZO‐1 (catalog no. 66452‐1; 1:100; ProteinTech Group) and anti-Claudin-1 (catalog no. 13050-1; 1:100; ProteinTech Group) primary antibody were applied for incubation at 4°C overnight followed by incubation of the corresponding secondary antibody (1:200) for an hour at room temperature, and then mounted with a medium containing DAPI for capturing pictures.
5
Epithelial-Mesenchymal Transition Protein Analysis
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Briefly, total protein was extracted using RIPA lysis buffer containing protease inhibitor, phenylmethylsulfonyl fluoride and phosphatase. BCA protein assay kit (Pierce, USA) was used to measure protein concentrations in samples. Approximately 10 μg protein was separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Proteins were then transferred to polyvinylidene difluoride membranes (Millipore; cat. no. ISEQ00010, USA). The membranes were incubated with the following primary antibodies: anti-E-cadherin (CST, USA), anti-Vimentin (CST, USA), anti-N-cadherin (CST, USA), anti-TGF-β1(CST, USA), anti-ZO-1(Proteintech, China), anti-IGFBP2 (Proteintech, China), anti-Occludin (Proteintech, China), anti-Claudin-1 (Proteintech, China), anti-FAK (CST, USA), anti-MEK1/2 (CST, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, China). ImageJ software was used for quantification.
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