Proteins were isolated with a lysis buffer composed of 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Triton X-100, 0.1% SDS and the Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltman, MA, USA). The BCA protein assay kit (Thermo Fisher Scientific) was used to quantify the protein content. An equal amount of protein from each sample was separated on CriterionTM Precast Gel Tris-HCl (Biorad, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA), as previously described55 (link). The membranes were blocked for 2 hours in 5% non-fat dry milk PBS with 0.1% Tween 20 (Sigma-Aldrich) at room temperature and incubated overnight at 4 °C with primary antibody. After washing, the membranes were incubated for 1 hour at room temperature with horseradish-peroxidase-conjugated secondary antibody. The following primary antibodies were performed: anti- Collagen IV (1:1000, Abcam), anti-Fibronectin (1:1000, Abcam), anti-Vitronectin (1:1000, Abcam), anti-CASP3 (1:1 000, Cell Signaling Technology), anti-CASP9 (1:500, Cell Signaling Technology), anti-BAX (1:1000 Cell Signaling Technology), anti-RHO (1:1000 Merck Millipore), anti-Vinculin (1:1000 Thermo Fisher Scientific). Vinculin was used as the loading control.
Anti collagen 4
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Anti-collagen IV is a laboratory reagent used for the detection and quantification of collagen IV in biological samples. It is a highly specific antibody that binds to collagen IV, a key structural component of basement membranes. This product can be used in various immunoassay techniques to measure collagen IV levels.
Automatically generated - may contain errors
Lab products found in correlation
Anti fibronectin, by Abcam (19 mentions)
Anti collagen 1, by Abcam (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Anti laminin, by Merck Group (4 mentions)
Anti e cadherin, by Cell Signaling Technology (4 mentions)
Anti α sma, by Abcam (4 mentions)
Triton x 100, by Merck Group (3 mentions)
Anti e cadherin, by Abcam (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti ctgf, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Anti α sma, by Merck Group (2 mentions)
Anti fibronectin, by Santa Cruz Biotechnology (2 mentions)
Anti collagen 4, by Affinity Biosciences (2 mentions)
Protease inhibitor, by Boster Bio (2 mentions)
Anti smad3, by Abcam (2 mentions)
Ripa buffer, by Solarbio (2 mentions)
Anti p smad3, by Abcam (2 mentions)
Anti tgf β1, by Abcam (2 mentions)
52 protocols using anti collagen 4
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of ECM Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After tissues were homogenized in liquid nitrogen, the homogenate was lysed on ice for 30 min in lysis buffer (BioTeKe, Beijing, China). The lysates (20–40 μg) of total protein were loaded per well and separated on a 10% SDS-polyacrylamide gel. Primary antibodies were anti-CTGF (1:5000 dilution, Abcam, Shanghai, China), anti-collagen-II (1:5000 dilution, Abcam), anti-collagen-IV (1:5000 dilution, Abcam), anti-Smad 2/3 (1:1000 dilution, Cell Signaling Technology, Boston, MA), anti-Smad 7 (1:1000 dilution, Santa Cruz Biotech, Santa Cruz, CA), anti-p-Smad2 (1:1000 dilution, Cell Signaling Technology), and anti-actin antibodies (1:5000 dilution, Santa Cruz Biotech). The secondary antibody was a peroxidase-coupled anti-goat IgG (GE Healthcare). The membrane was exposed to ECL Hyperfilm (GE Healthcare), and the film was developed. The bands were quantified by densitometry using ImageJ. Results were from triplicate experiments.
3
Croton Oil-Induced Vascular Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
croton oil experiments were performed as previously described (Schenkel et al., 2004 (link)). In brief, both male and female age- and sex-matched chimeric mice were exposed to 2% croton oil (Sigma-Aldrich) in a 4:1 mixture of acetone/olive oil (diluent) on one ear. The contralateral ear was treated with vehicle solution alone. After 6 h the animals were sacrificed. The ears were harvested and fixed in 4% paraformaldehyde overnight at 4°C. Tissue was permeabilized and blocked in a solution of 0.3% Triton X-100, 2.5% BSA, and 2.5% host species serum in PBS for 24 h at 4°C. Ears were then incubated with anti-PECAM (clone 2H8), anti–ICAM-1 (clone YN1/1.7.4; Abcam), anti–MRP-14 (Abcam), or anti–collagen IV (Abcam) in blocking solution overnight at 4°C. Tissue was then incubated with secondary Ab (Dylight550 goat anti–Armenian hamster, Alexa Fluor 488 goat anti–rat, or Alexa Fluor 647 goat anti–rabbit at 1:500) in PBS for 4 h at room temperature.
Stained tissue was mounted on glass slides, and postcapillary venules of 20–30 µm were imaged using an UltraVIEW VoX imaging system equipped with a CSU-1 spinning disk. Images were analyzed using Volocity software, which renders the optical sections into 3D images, thus allowing one to discern the position of leukocytes relative of the endothelium and basement membrane.
Stained tissue was mounted on glass slides, and postcapillary venules of 20–30 µm were imaged using an UltraVIEW VoX imaging system equipped with a CSU-1 spinning disk. Images were analyzed using Volocity software, which renders the optical sections into 3D images, thus allowing one to discern the position of leukocytes relative of the endothelium and basement membrane.
4
Protein Extraction and Western Blot Analysis of Renal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Renal cortex was homogenized and extracted in a cold buffer containing 0.1 mol/l Tris (hydroxymethyl) aminomethane HCl. The tissue extracts were then partially purified by ethanol extraction. Proteins from GENs or mesangial cells were isolated from using RIPA buffer (Thermo Scientific, USA). 50 μg of protein samples were isolated in 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, USA) by electroblotting. The membranes were blocked in 5% non-fat dried milk for 60 min at room temperature. For the detection of RhoA, the membrane and cytoplasm proteins were extracted from mesangial cells using Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology), then membrane and cytosolic proteins were separated on SDS-PAGE. The membranes were probed with first primary antibody anti-ETBR (Abcam, USA), anti-ETAR (Abcam, USA), anti-p-p65 (Invitrogen, USA), anti-p65 (Invitrogen, USA), anti-CTGF (Abcam, USA), anti-RhoA (Abcam, USA), anti-collagen IV (Abcam, USA), anti-Fibronectin (Abcam, USA), anti-p21 (Abcam, USA) and anti-β-actin (Invitrogen, USA) and incubated at 4°C overnight. After washing with PBST, membrane was cultivated with secondary antibody for 60 min at room temperature. β-actin was used as internal control.
5
Immunofluorescence Characterization of Cellular and Extracellular Matrix Components
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Both cellularized (with v-HCFs or a-HCFs) and decellularized samples were fixed in paraformaldehyde 4% in PBS (PFA, Alfa Aesar) for 15 min, washed with PBS, and cells were permeabilized with Triton X-100 (Sigma-Aldrich) 0.5% in PBS for 10 min. Samples were then blocked with bovine serum albumin (BSA, Sigma-Aldrich) 2% in PBS for 30 min, followed by staining with Phalloidin-Rhodamine (ThermoFisher) or primary and secondary antibodies, diluted in BSA 2% in PBS. Primary antibodies for fibroblasts staining were: Anti-Actin Smooth Muscle (α-SMA, Sigma Aldrich) and Anti-Discoidin Domain Receptor 2 (DDR2, ThermoFisher). Primary antibodies used for extracellular matrix protein detection were anti-Collagen I, anti-Collagen III, anti-Fibronectin, anti-Laminin, anti-Tenascin, (all purchased from Sigma-Aldrich), and anti-Collagen IV (Abcam). Secondary antibodies used were anti-mouse Alexa Fluor 555 and anti-rabbit Alexa Fluor 488 (both from ThermoFisher). Nuclei were counterstained with DAPI (Sigma-Aldrich). Samples were maintained in PBS during imaging by using Nikon Ti2-E fluorescence microscope (Nikon Instruments). Immunofluorescence experiments were performed in biological triplicate.
6
Multimarker Immunohistochemistry for Neurovascular Unit
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For single-labeling immunohistochemistry of cholinergic cell bodies and fibers, tissue sections were washed in 0.01 M PBS, blocked with 15% normal donkey serum (NDS; Sigma-Aldrich) and incubated overnight with either anti-ChAT (1:100) or anti-laminin (1:350, Sigma-Aldrich), after pre-treatment with pepsin (1 mg/mL in 0.2 N HCl, 30 s at 37°C). The next day, sections were washed in PBS and incubated for 2 h at room temperature with anti-donkey AlexaFluor 488 (Fisher Scientific, Loughborough, UK). For quadruple labeling of the NVU, sections were treated with pepsin, incubated overnight with anti-ChAT (1:100), washed in PBS and incubated with anti-goat AlexaFluor 555. After washing in PBS, sections were incubated simultaneously with anti-collagen IV (1:100, Abcam, Cambridge, UK), anti-α smooth muscle actin (α-SMA)-FITC (1:350, Sigma-Aldrich) and anti-glial fibrillary protein (GFAP, 1:500, Abcam). Sections were then developed with anti-rabbit AlexaFluor 405 and anti-chicken AlexaFluor 633 (1:200, Fisher Scientific). All fluorescent sections were coverslipped using Mowiol® (Sigma-Aldrich) containing 0.1% v/v Citifluor (Citifluor Limited, London, UK) mounting media.
7
Antibody Sources and Plasmid Construction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies were obtained from multiple sources: anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) and anti-AP1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-MBD2, anti-collagen I, anti-collagen IV, anti-TGF-β, anti-ERK1/2, anti-p-ERK1/2, and anti-fibronectin from Abcam (Cambridge, MA, USA); and anti-EGR1, anti-SMAD3, and anti-p-SMAD3 from Proteintech Group (Rosemont, IL, USA),NCM Universal Antibody Diluent (New Cell and Molecular Biotech,Suzhou, China). The recombinant human TGF-β1 was obtained from Proteintech Group. The plasmids containing the methylation promoter of EGR1 CpG-free pCpGI luciferase reporter, MBD2 and mtMBD2 (the deletion of the methylated DNA binding domain), were constructed by the Ruqi Biology (Guangdong, Guangzhou, China), according to previously published reports.28 (link),39 (link)
8
Immunohistochemical Analysis of Kidney Fibrosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed paraffin kidney sections were deparaffinised and incubated with the polyclonal primary antibody anti-FXR (1:50 dilution, Abcam Ltd, Cambridge), anti-fibronectin (dilution 1:1,000, Abcam) and anti-collagen IV (dilution 1:1,000, Abcam) overnight, followed by horseradish peroxidase anti-rabbit Envision system (Dako Cytochemistry, Tokyo, Japan) the following day. Staining was developed with 3.3diaminobenzidine tetrahydrochloride (Dako Cytochemistry, Tokyo, Japan) before counterstaining with Mayer’s haematoxylin stains. Antibody against rabbit IgG was used as a negative control. Images were analysed using an Olympus microscope (Olympus, Japan) and Image J software.
9
Quantifying Protein Abundances in Kidney Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The relative protein abundances in kidneys and HK-2 cells were analyzed using western blotting, as described previously (Xu et al., 2012 (link)). The following primary antibodies were used: anti-α-SMA (1:1,000, Sigma), anti-E-cadherin (1:1,000, Cell Signaling Technology), anti-vimentin (1:1,000, Santa Cruz Biotechnology), anti-collagen I (1:1,000, Abcam), anti-collagen IV (1:1,000, Abcam), anti-Wnt1 (1:100, Abcam), anti-Wnt4 (1:150, Santa Cruz Biotechnology), anti-Wnt3 (1:500, Abcam), anti-Wnt2b (1:700, Abcam), anti-Wnt7a (1:300, Abcam), anti-β-catenin (1:1,000, Cell Signaling Technology), anti-Snail 1 (1:1,000, Santa Cruz Biotechnology), anti-PAI-1 (1:1,000, Santa Cruz Biotechnology), and anti-NF-κB p65 (1:1,000, Cell Signal). Horseradish peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse or anti-goat IgG, 1:5,000, Jackson ImmunoResearch) were used according to the manufacturer’s instructions. Image analysis software (Image J, National Institutes of Health) was used to determine the gray value of all bands.
10
Immunohistochemical Analysis of CCL-2, CCL-5, α-SMA, and Collagen IV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry was performed as previously described in paraffin-embedded tissue sections at a thickness of 4 µm [18] (link). The primary antibodies used in this study were as follows: anti-CCL-2 (1∶200, Abcam), anti-CCL-5 (1∶100, Santa Cruz), anti-α-SMA (1∶100, Sigma) and anti-collagen IV (1∶400, Abcam). Sections were counterstained with hematoxylin. Positive staining were quantified by Image Pro Plus Software 5.0 (Media Cybernetics, Silver Spring, USA) and presented as IOD value.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!