Histological analysis of fibrosis was carried out on fixed renal tissue, embedded in paraffin, and sectioned at a thickness of 4 μm. Connective tissue deposition was examined with Picrosirius Red Stain Kit (Polysciences, Inc, Warrington, PA, USA) and collagen I (1:100, Millipore, Billerica, MA, USA), staining, as we have previously described [69 (link)-71 (link)]. Picrosirius Red-stained sections were photographed under polarized light to achieve maximal brightness and the percentage of positive interstitial staining was quantified. For collagen I staining fluorescent images were collected with Leica DFC310 FX camera. At least 10 images of both cortex and medulla were analyzed. Image J image analysis software was used to quantify the percentage of tissue fibrosis in the cortex and medulla [69 (link)-71 (link)]. Greyscale images were subjected to threshold analysis producing the value of pixel intensity representing positive staining and then expressed as a percentage of the value of pixel intensity of the whole image. Additionally, anti-collagen III (1:100; Abcam, Cambridge, MA, USA) staining and double staining for collagen I and αSMA (1:10.000; Sigma, Saint Louis, MI, USA) was performed to assess specifically contribution of different types of collagen to fibrosis in a qualitative manner.
Collagen 1
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, China
Collagen I is a type of extracellular matrix protein that is a key structural component of various tissues, including skin, bone, and connective tissue. It is commonly used in research and laboratory applications to study cell-matrix interactions, tissue engineering, and other biomedical applications.
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Lab products found in correlation
Fibronectin, by Merck Group (36 mentions)
Laminin, by Merck Group (17 mentions)
Collagen 4, by Merck Group (15 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (13 mentions)
Bovine serum albumin (bsa), by Merck Group (12 mentions)
Vitronectin, by Merck Group (6 mentions)
Crystal violet, by Merck Group (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
α sma, by Merck Group (4 mentions)
Matrigel, by Corning (4 mentions)
Phosphate buffered saline (pbs), by Merck Group (4 mentions)
Matrigel, by BD (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
α sma, by Abcam (3 mentions)
Fibronectin, by Thermo Fisher Scientific (3 mentions)
Sulfo sanpah, by Thermo Fisher Scientific (3 mentions)
Picrosirius red stain kit, by Polysciences (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
Transwell plate, by Corning (3 mentions)
195 protocols using collagen 1
1
Histological Evaluation of Renal Fibrosis
2
Functionalization of PEG-NHS Hydrogel Scaffolds
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A similar protocol to that described above was used to produce an N-hydroxysuccinimide (NHS) ester-functionalized ICC scaffold, with a pre-polymer solution containing 50% (w/v) PEG-DA, 10% (w/v) acryloyl-PEG-NHS (Laysan Bio, AL), and 0.05% photo initiator in deionized water. The prepared PEG-NHS ICC scaffold was coated with either collagen I or fibronectin by centrifugation, shaking, and incubation in 20 μg/ml collagen I (Merck Millipore, Germany) or fibronectin (Merck Millipore) solution in pH 7.4 PBS at 4 °C overnight. Excess collagen I or fibronectin was removed by repeated washing with PBS.
To characterize the ECM coating, the ICC scaffolds were fixed in 4% paraformaldehyde (Alfa Aesar, MA) then incubated with a mouse primary antibody against collagen I or fibronectin (Abcam, UK) at 4 °C overnight in the presence of 3% bovine serum albumin (BSA; Sigma-Aldrich) and labeled with an anti-mouse secondary antibody conjugated to a fluorophore. Green fluorescent Alexa Fluor 488 was used to label collagen I and red fluorescent Alexa Fluor 555 was used to label fibronectin. After PBS washing, the ECM-functionalized scaffolds were imaged using a Carl Zeiss LSM 710 confocal microscope.
To characterize the ECM coating, the ICC scaffolds were fixed in 4% paraformaldehyde (Alfa Aesar, MA) then incubated with a mouse primary antibody against collagen I or fibronectin (Abcam, UK) at 4 °C overnight in the presence of 3% bovine serum albumin (BSA; Sigma-Aldrich) and labeled with an anti-mouse secondary antibody conjugated to a fluorophore. Green fluorescent Alexa Fluor 488 was used to label collagen I and red fluorescent Alexa Fluor 555 was used to label fibronectin. After PBS washing, the ECM-functionalized scaffolds were imaged using a Carl Zeiss LSM 710 confocal microscope.
3
Characterization of Mechanotransductive Pathways in Fibroblasts
4
Co-Culture Model for Blood-Brain Barrier
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We used a co-cultivation model including hCMEC/D3 cells and astrocytes seeded on each side of a porous insert allowing cell-cell contacts as previously described [9 (link), 17 ]. Briefly, cell culture inserts for 24-well plates with 3.0-μm pore translucent PET membrane (Corning Life Sciences) were coated with 150 μg/mL collagen-1 (Sigma-Aldrich). Astrocytes were then seeded on the basal side of the membrane and incubated for 4 h before hCMEC/D3 cells were seeded on the upper side of the membrane. Cells were allowed to grow in 150 μL (upper chamber) and 750 μL (collector) of BBB medium, composed of 50% complete EBM-2 and 50% DMEM supplemented with 10% FBS, during 6 days to reach confluence. BBB integrity was assessed by measuring permeability to dextran-rhodamine. The culture medium in the upper chamber was replaced with BBB medium supplemented with 1 mg/mL 70-kDa dextran-rhodamine (Life Technologies). After 6 h, the fluorescence intensity in collectors was measured using a Varioskan flash multi-mode reader (Thermo Fisher Scientific). Samples displaying dextran-rhodamine permeability superior to 20% of the empty control were discarded.
5
Analyzing Mitochondrial Dynamics in Metabolism
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The reagents and antibodies used in this study were obtained from the indicated suppliers: succinic acid, N-acetyl-L-cysteine (NAC) from Sigma (St. Louis, MO, USA); gemigliptin from LG Chem (Seoul, Korea); primary antibodies: ERK1/2, p-ERK1/2, DRP1, p-DRP1 (Serine 616), MFF, and p-MFF from Cell Signaling Technology (Richmond, CA, USA); p-DRP1 (Serine 616) from Invitrogen (Waltham, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from GeneTex (Irvine, CA, USA), GPR91 from Santa Cruz Biotechnology (Dallas, TX, USA), α-SMA from Abcam (Cambridge, England), and collagen 1 from Sigma-Aldrich (St. Louis, MO, USA).
6
Cell Migration and Invasion Assay
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Cells were plated in the upper chamber of an insert (Corning-Costar, Lowell, MA, USA) coated with or without collagen 1 (Sigma-Aldrich, St. Louis, MO, USA) containing 1% FBS-media for cell invasion or cell migration assays, respectively. The bottom chamber contained 10% FBS-media as a chemoattractant. After 8 to 10 h of incubation, cells and medium were removed from the upper chamber and migrated or invaded cells were fixed with methanol and stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO, USA). Images were taken at ×100 magnification under a bright-field microscope (Olympus CKX41, Tokyo, Japan). Quantification was performed under a light microscope with × 200 magnification by counting migrated or invaded cell number in five random fields per chamber.
7
3D Breast Cancer Tumor Spheroid Culture
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Breast cancer cells (3.3x105) were resuspended in 2 ml of DMEM and aggregated over agar (2h at 37°C, 5% CO2). Aggregates were centrifuged at 500 rpm (3 min) and resuspended in 1 ml of rat tail collagen I (BD Biosciences, Franklin Lakes, New Jersey, US). This was adjusted to 1 mg/ml by diluting with Dulbecco’s PBS. Eight parts of collagen 1 were mixed with one part 10x Hank’s buffered salt solution (Sigma-Aldrich, St Louis, Missouri, US). Sodium hydroxide was added drop wise to neutralise the pH. One volume of DMEM was added and mixed into the collagen 1 solution by gentle pipetting. One hundred μl of the gel mixture was pipetted onto a 10mm Petri dish (MatTek Corp, Ashland, Massachusetts, US), left to solidify (10 min 37°C, 5% CO2) before covering with 1.5 ml complete DMEM. Gels were cultured for 7 days, with media replaced every 2–3 days.
8
Cell Culture of Oral Cancer Cell Lines
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This studydid not require any human/animal subjects to acquire any ethical approval. The adenoid squamous carcinoma cell line (TYS), derived from a minor salivary gland, was a kind gift from Dr. Koji Harada, University of Tokushima, Japan. Normal oral mucosal fibroblasts (MM1) and oral cancer-associated fibroblasts (COM D24) were isolated in-house from explant cultures of biopsies from the Oral Surgery Clinic, Ninewells Hospital, Dundee Cells were cultured and maintained in minimum essential medium (MEM) supplemented with 10% (v/v) foetal calf serum and 200 mM glutamine and incubated at 37 °C in a humidified incubator with 5% CO2. Prior to growth of the MM1 and COM D24 cell lines, culture dishes were coated with collagen 1 (#C-3867, Sigma, St. Louis, MO, USA).
9
Collagen Gel Assay for Cell Migration
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Collagen gels (2 mg/mL) were made by mixing collagen 1 (#C-4243, Sigma, St. Louis, MO, USA) with 10XMEM medium and 7.5% (w/v) sodium bicarbonate and incubated for 1 h to allow complete polymerisation. Then, TYS cells were plated on the top of the gels at a density of 2 × 104 cells/well in a 48-well plate, and the plate was then incubated for 4 h to allow cell attachment. After this, the medium was discarded and conditioned medium with or without the inhibitors was added in the wells and incubated for 48 h. Serum-free MEM was added to some wells and regarded as the negative control. Five areas were chosen randomly inside the gel in each well and pictures were taken of the migrated cells. The mean number of migrated cells per well was calculated and the results were compared to the negative control. The experiments were carried out three times.
10
Isolation of Primary Murine Hepatocytes
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Primary hepatocytes were isolated from 12-week-old C57BL/6J mice as described in detail previously [24 ]. The cells were plated at 80% confluency on collagen-coated 6-well plates. Collagen-coated plates were prepared at 6.25 μg/cm2 using collagen 1 (Cat#: C3867, Sigma), according to the manufacturer's protocol. Seeded plates were left in the incubator at 37 °C and 5% CO2 for 5 h to allow the cells to attach, then each well was gently washed with 3 mL of Waymouth medium (Cat#: W1625, Sigma) to remove unattached cells and cultured overnight. The medium was then replaced with MEM alpha as indicated above.
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