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31 protocols using anti collagen 3

1

Achilles Tendon Injury and Repair Dynamics

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Animals were euthanized with anesthetic overdose. Achilles tendons were isolated from surrounding tissue and dissected from both sides of hind limbs. The proximal ends of the Achilles tendons were disconnected at the end of the gastrocnemius, plantaris, and soleus, and the distal ends at the calcaneus. Left leg tendons of eight control rats and six burn rats for each time point (1, 3, 7, and 14 days) were stored in RNAlater (Qiagen), whereas right leg tendons were snap frozen for protein extraction. Samples were stored in −80°C for further analysis. Tendon RNA was obtained with RNAeasy Universal kit (Qiagen). cDNA conversion was done with iScript kit (Bio-Rad), and qPCR with SsoFast Eva Green kit (Bio-Rad). Primers for IL-6, TNF, IL-1β, col1a1, col3a1, MMP9, MMP13, and TGFβ1 were purchased from QuantiTect Primers (Qiagen). Gene expression was calculated using the ΔΔCt method with 18 s as housekeeping.
For protein extraction, tendon tissue was homogenized in RIPA buffer (Invitrogen) with proteinase inhibitor cocktail (Sigma). Protein quantification was performed with BCA assay (Pierce); 10 μg of total protein was used for Western blot. Antibodies included anti-collagen I (Abcam), anti-collagen III (Abcam), goat anti-mouse HRP (Pierce), and goat anti-rabbit HRP (Pierce), which were prepared in 2% BSA. Blots were developed with ECL (Pierce) using a CCD camera system.
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2

Immunohistochemical Evaluation of Wound Biomarkers

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After dewaxing, hydration, endogenous peroxidase activity, and nonspecific binding blocking treatment, the sections were blocked with 5% BSA at 37°C for half an hour and at 4°C overnight incubated with primary antibody: Anti-FP receptor (Cayman 101802), Anti-CollagenI (Abcam ab34710), Anti-CollagenIII (Abcam ab7778), Anti-MMP2 (Abcam ab97779), and Anti-MMP9 (Abcam ab76003). The slices were washed three times with PBS and incubated with secondary antibodies at 37°C for 50 minutes. The bound secondary antibodies was detected using DAB solution (Zhongshan Jinqiao Biotechnology, Beijing, China). The reactions were observed under a microscope and stopped in distilled water when target area turned yellow. Lastly, the nuclei were stained with hematoxylin, and the slides were dehydrated, cleared, and mounted. The images were acquired using an Olympus DP72 digital imaging system and analyzed using Image Pro Plus (IPP) 6.0 software.
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3

Hepatic Protein Expression Analysis

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Hepatic tissues were dissected and immediately frozen in liquid N2. AML-12 cells extracts were prepared in ice-cold buffer (1% Triton X-100, 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerin, 10 mM Na4P2O7, 20 mM glycerophosphate, 10 mM NaF, 10 mM sodium orthovanadate and proteinase inhibitor mixture) until the cells were completely lysed. The protein concentrations were measured using a bicinchoninic acid protein assay kit (Sigma-Aldrich, USA). Total protein (40 μg) was resolved on 8%-12% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes (Millipore, USA). Blotted membranes were then incubated by either anti-ERK (1:1000, CST), anti-phospho-ERK (1:2000, CST), anti-PPARα (1:200, Abcam), anti-phosphoPPARα (Ser12) (1:1000, Abcam), anti-Collagen I (1:1000, Abcam), anti-Collagen III (1:2000, Abcam), or β-tubulin (1:1000, CST) antibodies. After several washes, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000, Sigma-Aldrich) or anti-goat IgG (1:5000, Signalway Antibody).
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4

Western Blot Analysis of Cellular Proteins

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The procedure of western blot was the same as the previous paper [23 (link)]. In brief, the total protein from the cells was lysed with radio immunoprecipitation assay buffer (Solarbio Biotech). Protein concentrations were measured using BCA protein assay kit (Beyotime) according to the manufacturer’s instructions. Then, equivalent amount of protein were separated by SDS-PAGE and transferred into polyvinylidene fluoride membranes (Bio-Rad). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight and then incubated with secondary antibodies for 2 h at room temperature. Bands were visualized with an ECL detection kit (Thermofisher) using ChemiDoc XRS Plus (BioRad) and analyzed using the Image J software. All primary antibodies and secondary antibodies in the study are listed as follows: anti-ACSM5 antibody (1:500, Proteintech), anti-collagen I (1:1000, Abcam), anti-collagen III (1:1000, Abcam), anti-MMP2 (1:1000, Proteintech), anti-MMP13 (1:1000, Proteintech), and anti-β-actin antibody (1:5000, Abcam).
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5

Western Blot Analysis of Cardiovascular Markers

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Western blots were performed using previously described methods 18. The blots were incubated with the following specific primary antibodies overnight at 4°C on a shaker: anti‐C/EBPβ (Abcam, Cambridge, MA, USA and Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐ACE2, anti‐ACE, anti‐AT1R, anti‐AT2R, anti‐TGFβ1, anti‐MMP2, anti‐MMP9, anti‐collagen I, anti‐collagen III (all from Abcam), and B‐cell lymphoma/leukaemia‐2 (Bcl‐2), anti‐Bcl2‐associated X protein (Bax) and anti‐MasR (all Cell Signalling Technology, Boston, MA, USA). All protein levels were normalized to β‐tubulin (Proteintech Group, Wuhan, Hubei, China). Bands were detected using an Amersham Imager 600 (Fairfield, CT, USA) and were quantified with Photoshop CS6 (San Jose, CA, USA).
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6

Western Blot Analysis of Kidney Cell Markers

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HK-2 cells or renal tissue samples were rinsed with ice-cold PBS and lysed using RIPA lysis buffer (Beyotime, Shanghai, China). The lysates were centrifuged at 12,000×g at 4°C for 10 min, and the supernatants were collected to measure protein concentration by BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of proteins (30 μg) were separated by 12% SDS-PAGE and then electrophoretically transferred to a PVDF membrane (Beyotime, Shanghai, China). The membranes were blocked with 5% skim milk in TBST and then incubated with primary antibodies (anti-CK-18: Abcam, 1:1000; anti-α-SMA: Abcam, 1:1000; anti-MMP9: Abcam, 1:1000; anti-p-Smad2: Abcam, 1:1000; anti-p-Smad3: Abcam, 1:1000; anti-Collagen III: Abcam, 1:1000; anti-Fibronectin: Abcam, 1:1000; anti-β-actin: Abcam, 1:1000; Cambridge, MA, USA) at 4°C overnight. Then, membranes were incubated with secondary antibodies (Goat Anti-Rabbit IgG: Abcam, 1:5000, Cambridge, MA, USA) at room temperature for 1 h. The bands of proteins were visualized using a chemiluminescent detection system (BeyoECL Plus, Beyotime, Shanghai, China). Finally, band densities were examined using IPP Image-Pro Plus software. β-actin was used as the internal control.
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7

Quantitative Protein Expression Analysis

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Collected whole cellular proteins and then determined the protein concentrations by BCA. Next, 45 μg proteins was resolved over 10% polyacrylamide gels and then transfer-embedded onto a PVDF membrane (Solarbio, Beijing, China). Blocking of the membrane was done in 5% non-fat milk for two hours at RT and then overnight inoculated with the appropriate primary antibodies at 4° C. For PI3K/AKT/mTOR pathway, anti-PI3K (ABclonal; 1:1000), anti-AKT (ABclonal; 1:1000), anti-p-AKT (Abcam; 1:1000), anti-mTOR (Abcam; 1:1000) and anti-p-mTOR (Abcam; 1:1000) antibodies were used. For cellular apoptosis, anti-c-PARP (1:1000, Proteintech), anti-caspase-3 (Proteintech, Wuhan, China; 1:1000) and anti-caspase-8 (Proteintech; 1:1000) antibodies were used. For EMT-related proteins, anti-N-cadherin (Abcam; 1:1000), anti-E-cadherin (ProteinTech; 1:1000), anti-α-SMA (Affinity Biosciences; 1:1000), anti-Vimentin (Affinity Biosciences; 1:1000), anti-Collagen I (Abcam; 1:1000) and anti-Collagen III (Abcam; 1:1000) were used. GADPH (Bioworld; 1:1000) was employed as the internal reference. After rinsing thrice in TBST, incubated the membranes with anti-rabbit secondary antibody (Proteintech; 1:5000) for one hour at RT. Lastly, the protein bands were rinsed five times in TBST and visualization via chemiluminescence detection done on the autoradiographic film.
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8

Quantitative Western Blot Analysis of Cardiac Fibrosis

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Western blot analysis was performed as described by us (Wang et al., 2019b (link)). Briefly, proteins was extracted from CFs with RIPA lysis buffer (Biosharp, Hefie, China) and quantified with a BCA protein assay reagent (Beyotime, Haimen, China). Equal of protein lysates was loaded into per lane for SDS-PAGE detection before transferred to PVDF membranes (Millipore, Billerica, MA, USA). Next, all membranes were blocked with 5% nonfat milk solution in TBST buffer for 1 h at room temperature and then incubated with primary antibodies on a shaker at 4 °C overnight. The primary anti-bodies: Anti-Collagen I (Cell Signaling Technology, 91144, dilution: 1:1,000), Anti-Collagen III (Abcam, ab7778, dilution: 1:7,000), Anti-α-SMA (Cell Signaling Technology, 19245, dilution: 1:1,000), Anti-p-Smad2 (Cell Signaling Technology, 18338, dilution: 1:1,000), Anti-p-Smad3 (Cell Signaling Technology, 9520, dilution: 1:1,000), Anti-TGF-β2 (Bioss, bs-20412R, dilution: 1:1,000) and Anti-β-actin Antibody (Abcam, ab8227, dilution: 1:1,000), were used. The next day, membranes were incubated with secondary antibody (Proteintech, SA00001-2, dilution: 1:5,000) for 1 h at room temperature. And then the membranes were detected with ECL chemiluminescent substrate (Biosharp, Hefie, China). β-actin acted as the internal standard.
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9

Protein Extraction and Western Blot Analysis

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RIPA buffer was used to extract total protein from HUFs, human and rat urethral tissues. BCA protein assay (Thermo Fisher Scientific) was performed to detect the protein concentration. Equal amounts of protein (20 µg) were separated by 10% SDS-PAGE gels and transferred to PVDF membrane (Millipore, USA). Next, the membranes were blocked in 5% fat-free milk for 2 h, and incubated with the following antibodies at 4 °C overnight: anti-Wnt3a (1:1000, Abcam), anti-β-catenin (1:1000, Abcam), anti-DKK1(1:1000, Abcam), anti-collagen I (1:1000, Abcam), anti-collagen III (1:1000, Abcam), anti-α-SMA (1:1000, Abcam), anti-p-GSK-3β (1:1000, Abcam), anti-GSK-3β(1:1000, Abcam), anti-p-Smad2(1:1000, Abcam), anti-p-Smad3 (1:1000, Abcam), anti-Smad2/3 (1:1000, Abcam) and anti-GAPDH (1:5000, Abcam, Cambridge, UK). Membranes were cut horizontally based on the differences of molecular weight. Then membranes washed with TBST (Beyotime Biotechnology, Shanghai, China) and incubated with corresponding secondary antibodies (goat anti-rabbit IgG H&L (HRP), 1:10000; rabbit anti mouse IgG H&L (HRP), 1:10000; Abcam, USA) at room temperature for 1 h. Additionally, membranes were stripped with Western Blot Stripping Buffer (Cell Signaling Technology), and re-probed with target proteins. Target blots signals were observed by an enhanced chemiluminescence-detecting kit (Thermo Fisher, MA, USA).
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10

Collagen and SMA Expression Analysis

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HFBs after different treatments were washed with ice-cold PBS three times. 80 μL RIPA buffer containing protease and phosphatase inhibitor was added to lyse the cells and centrifugated for 10 min (14,000×g) at 4 °C. 50 mg total protein determined by BCA was processed by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat milk and incubated with different antibodies anti-Collagen III (1:1000, Abcam, UK) anti-Collagen I (1:1000, Abcam, UK), anti-SMA (1:1000, CST, USA). The membranes were imagined with ECL detection system (Alpha Innotech, San Leandro, CA).
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