NRK-52E cells were lysed using the protein lysis buffer radio immunoprecipitation assay containing a 50× protease inhibitor. Equal amounts of total extracted protein were electrophoresed on a 12% SDS-PAGE gel and transblotted onto 0.2 μm polyvinylidene fluoride membranes. After being blocked with 5% non-fat milk powder in tris-buffered saline and 0.1% Tween-20, the membranes were incubated with primary antibodies against NLRP3 (Abcam, 1:1000), high-mobility group box 1 (HMGB1) (CST, 1:1000), alpha-smooth muscle actin (alpha-SMA) (CST, 1:1000), CollagenI (Affinity, 1:1000), IL-1beta (Abcam, 1:1000), GAPDH (Boster, 1:4000), and beta-actin (Boster, 1:1000), followed by the addition of horseradish peroxidase (HRP)-labeled secondary antibodies. Immune-reactive band signals were detected using an enhanced chemiluminescence Western blotting system, and band intensity was measured using ImageJ software.
Collagen 1
Manufactured by Affinity Biosciences
Sourced in China, United States
Collagen I is a type of collagen that is a major structural component of the extracellular matrix in various tissues. It is the most abundant collagen found in the human body. Collagen I provides mechanical strength and support to tissues such as skin, bone, tendon, and ligament.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Affinity Biosciences (2 mentions)
α sma, by Abcam (2 mentions)
Beta actin, by Boster Bio (1 mentions)
Il 1 beta, by Abcam (1 mentions)
Gapdh, by Boster Bio (1 mentions)
Tgf β, by Affinity Biosciences (1 mentions)
Mmp 2, by Affinity Biosciences (1 mentions)
Mmp 9, by Affinity Biosciences (1 mentions)
Nf κb, by Affinity Biosciences (1 mentions)
Socs1, by ABclonal (1 mentions)
Alpha smooth muscle actin α sma, by Affinity Biosciences (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Proteintech (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bicinchoninic acid protein assay kit, by Solarbio (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Solarbio (1 mentions)
Loxl2, by Abcam (1 mentions)
E cadherin, by Affinity Biosciences (1 mentions)
Vimentin, by Affinity Biosciences (1 mentions)
Enhanced chemiluminescence substrate, by Merck Group (1 mentions)
Iseq00010, by Merck Group (1 mentions)
11 protocols using collagen 1
1
Western Blot Analysis of NLRP3 and Fibrosis Markers
2
Immunofluorescence Analysis of Lung Cells
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The different groups of BEAS-2B and HFL-1 cell lines were used to prepare the paraffin slides. After dewaxing and dehydrating, antigen retrieval was performed at 95 °C for 10 min. The paraffin slices were blocked with 5% BSA at room temperature for 1 h. For immunofluorescence, FITC-labeled fluorescent primary antibody collagen I (Affinity, Jiangsu, China, dilution: 1:200) and α-SMA (CST, Boston, USA, dilution: 1:200) were incubated overnight at 4 °C. On the next day, sections were rewarmed, washed with PBS, and antirabbit IgG (H + L) Alexa Fluor® 488 conjugate secondary antibody (CST, Boston, USA, dilution: 1:500) was incubated in the dark for 1 h. After restaining with DAPI, the slices were observed with a fluorescence microscope (OLYMPUS, Japan, BX51) and photographed. The percentage of fluorescently labeled cells in rat lung tissues was counted with different fluorescently labeled cell-counting methods and the fluorescence intensity statistics of Image J. In addition, we anonymously shuffled the image order statistics to prevent statistical preference. Three fields of view were also analyzed. The magnification was 100×.
3
Immunohistochemical Analysis of Wound Tissue
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The available wound tissue sections were deparaffinized and rehydrated. Subsequently, 3% H2O2 in methanol was used for 10 min to block endogenous peroxidase activity. After boiling in 0.01 mol/L citric acid solution for 20 min to retrieve the antigen, normal goat serum was applied at 37 °C for 10 min. The sections were treated with the following primary antibodies: collagen I (Affinity, Changzhou, China), NF-κB (Affinity, Changzhou, China), MMP-2 (Affinity, Changzhou, China), MMP-9 (Affinity, Changzhou, China), and TGF-β (Affinity, Changzhou, China). After incubation for 1 h, sections were washed with phosphate-buffered saline (PBS) and incubated with biotinylated goat anti-mouse IgG antibody (Zhongshan Biology Technology Co., Ltd., Beijing, China) at 37 °C for 30 min. Following staining with 3, 3'-diaminobenzidine (DAB)/H2O2 and HE, sections were cleared and mounted for observation and analysis.
4
Western Blot Analysis of Lung Proteins
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Total protein was extracted from lung tissues exposed to OVA by lysis solution (mixed with 1% phenylmethylsulfonyl fluoride) and was quantified by using a Bicinchoninic Acid Protein Assay Kit (Solarbio, Beijing, China). 20 μg protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by a transfer to polyvinylidene fluoride membranes (Millipore). Next, membranes were blocked with 5% (M/V) skimmed milk for an hour and were incubated with primary antibodies including alpha-smooth muscle actin (α-SMA; Affinity, Changzhou, China), collagen I (Affinity), suppressor of cytokine signaling 1 (SOCS1; ABclonal Biotechnology, Shanghai, China), p38 (ABclonal Biotechnology), and p-p38 (ABclonal Biotechnology) at 4°C overnight. Glyceraldehyde-3-phosphate dehydrogenase (Proteintech. Wuhan, China) was used as a loading control. All primary antibodies were diluted 1:1000 except for Glyceraldehyde-3-phosphate dehydrogenase (diluted 1:10,000). Subsequently, protein blots were incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1:3000; Solarbio) at 37°C for an hour. The relative protein levels were visualized by employing horseradish peroxidase chromogenic substrate (Solarbio). The images were captured by WD-9413B gel imaging system (Liuyi Biotechnology, Beijing, China).
5
Western Blot Analysis of EMT Markers
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Harvested cells and tumor tissues were lysed, and the protein concentration was determined using bicinchoninic acid assay. The lysates were separated by electrophoresis and then transferred onto polyvinylidene difluoride membranes (CAT: ISEQ00010, Millipore). The membranes were blocked and then incubated with primary antibodies of LOXL2 (CAT: ab96233, Abcam, UK), E-cadherin (CAT: AF0131, Affinity, China), Vimentin (CAT: AF7013, Affinity, China) and Collagen I (CAT: AF7001, Affinity, China). GAPDH (CAT: 5174, CST, USA) was used as the loading control. After 2 h, the membranes were incubated with a horseradish peroxidase-labelled secondary antibody. Protein expression was assessed by enhanced chemiluminescence substrate (Millipore).
6
Immunofluorescence Staining of Cell Markers
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Immunofluorescence staining was conducted as previously (39 (link)). After fixation with cold methanol: acetone (1:1), the cells were incubated with a blocking solution containing 10% goat serum, followed by overnight incubation with primary antibodies, and finally with secondary antibodies at 37°C for 1 hour. Primary antibodies for immunofluorescence included those for p65 (8242, Cell Signaling Technology), Collagen I (AF7001, Affinity), p-p65 (3033, Cell Signaling Technology), SIRT1 (8469, Cell Signaling Technology). Cy3-labeled goat anti-rabbit IgG and FITC-labeled goat anti-rabbit IgG were used as secondary antibodies. The cells were counter-stained with Hoechst to show the nucleus and examined on a Zeiss LSM780 confocal microscope (Oberkochen, Germany).
7
Investigating Fibrosis Pathways in TGF-β1 Model
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Deg-AZM (≥98.0%) was manufactured by chemical laboratory, College of Pharmacy, Nankai University. Recombinant Human TGF-β1 was purchased from PEPROTECH (USA). Antibodies to α-SMA, Collagen I, Fibronectin, P-Smad3/Smad3, P-Akt/Akt, P-ERK/ERK, P-P38/P38 were from Affinity Biosciences (Cincinnati, OH, USA). GAPDH, E-cadherin, Vimentin, N-cadherin antibodies were from Cell Signaling Technology (Danvers, MA, USA). Goat pAb to Rb IgG (HRP) and Rb pAb to Ms IgG were from ImmunoWay (Shanghai, China). TRIzol reagent was acquired from Ambion Life Technology (Shanghai, China). DEPC Treated H2O and Rnase Away H2O were purchased from Life Technologies. UNICON® Qpcr SYBR Green Master Mix was from YEVSEN (Shanghai, China). BLM was from Nippon Kayaku (Tokyo, Japan). Hematoxylin-eosin solution and Masson’s Trichrome Stain Kit were from Solarbio.
8
Liver Tissue Histopathological Analysis
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The livers were fixed with 4% paraformaldehyde for 48 h, embedded in paraffin, and sliced into 4 μm sections. Then, the sections were stained with H&E for the morphological evaluation and Masson stain or Sirius-red stain for the assessment of collagen. The collagen I (1:50, Affinity) and α-SMA (1:200, Abcam) immunohistochemical staining was performed following heat-induced epitope retrieval as previously described18 (link).
9
Immunofluorescence analysis of IMR90 cells
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IMR90 cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 20 min and then blocked with 1% BSA for 30 min. Cells were incubated with α-SMA and Collagen I were visualized with an overnight with specific fluorochrome primary antibodies including α-SMA (Abcam, United States), Collagen I (Affinity, China) at a concentration of 1:100. After extensive washing with PBS, cells were incubated with goat Alexa Fluor 488-labeled secondary antibody (Life Technologies, United States) for 1 h at room temperature and nuclei were stained with DAPI. The images were obtained by using Olympus FluoView® FV1200 confocal laser scanning microscope (Olympus Corporation, Center Valley, PA).
10
Kidney Protein Extraction and Analysis
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Kidney tissues of rats from each group were harvested. Proteins in kidney tissues and cells were extracted using radioimmunoprecipitation assay lysis buffer (Solarbio, Beijing, China) containing 1% phenylmethanesulfonyl fluoride (Solarbio) on ice. After determination of protein concentration with a BCA Assay Kit (Solarbio), the total protein lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidenedifluoride (PVDF) membranes (Millipore Corp, Darmstadt, Germany). Nonspecific binding sites were blocked with 5% skim milk. Then, the PVDF membranes were incubated overnight with the following primary antibodies: fibronectin (1:500; Affinity Biosciences), α-smooth muscle actin (α-SMA) (1:500; Affinity Biosciences), collagen I (1:1,000; Affinity Biosciences), collagen III (1:1,000; Affinity Biosciences), NLRP3 (1:500; Affinity Biosciences), cleaved caspase-1 (1:1,000; Affinity Biosciences), and GAPDH (1:5,000; Solarbio Life Science) at 4°C, followed by incubation at 37°C for 60 min with HRP-conjugated secondary antibodies (1:3,000; Solarbio). The membranes were visualized using an Enhanced Chemiluminescent Substrate Kit (Solarbio).
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