The frozen sections were fixed in cold acetone for 15 minutes. After a protein block, immunofluorescent staining of the endothelial cells or basement membrane was performed using antibodies to CD31 (BD Biosciences, San Jose, CA), type-IV collagen (Abcam, Cambridge, MA), or Ki67 (Abcam), respectively [26 ]. The sections were then incubated with the corresponding fluorochrome-labeled secondary antibodies (Jackson Immunoresearch). The images were captured using a confocal laser-scanning microscope (Carl Zeiss MicroImaging Inc., Thornwood, NY) and analyzed using the built-in image-analysis software [26 ]. The ruby-red fluorescence of anthracyclines in the tumor tissue was also imaged using confocal microscopy at the excitation and emission wavelengths of 488 nm and 590 nm, respectively [27 (link), 28 (link)]. The imaged fluorescence of DOX originates mostly from free DOX, because the solid state of DOX inside the PLD strongly quenches the fluorescence of liposomal DOX [29 ].
Type 4 collagen
Manufactured by Abcam
Sourced in United States, United Kingdom
Type IV collagen is a structural component of basement membranes. It is a major constituent of the extracellular matrix and provides mechanical support to various tissues.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Abcam (4 mentions)
Laminin, by Abcam (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Type 3 collagen, by Abcam (2 mentions)
α sma, by Abcam (2 mentions)
Type 1 collagen, by Abcam (2 mentions)
β actin, by Merck Group (2 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Fluorochrome labeled secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Wnt3a, by R&D Systems (1 mentions)
Versican, by Abcam (1 mentions)
P gsk 3β, by Abcam (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
P akt, by Abcam (1 mentions)
P erk, by Abcam (1 mentions)
β catenin, by Abcam (1 mentions)
Molecular imager chemidoc xrs, by Bio-Rad (1 mentions)
17 protocols using type 4 collagen
1
Analyzing Anthracycline Fluorescence in Tumor Tissue
2
Immunoblotting Quantification of ECM and Signaling Proteins
3
Western Blot Analysis of Cell Proteins
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Cells were washed with PBS, extracted with sample buffer (pH 6.8) consisting of 5% 2-mercaptoethanol, 10% glycerol, and 0.1 mg/mL bromophenol blue dissolved in Tris-HCl, and boiled at 100 °C for 5 min. To load the same amount of protein, BCA assay was performed, and the same amount of protein (20–40 μg) was separated via electrophoresis, on 8–10% SDS-PAGE, and transferred onto a nitrocellulose membrane. Subsequently, these separated proteins were incubated with primary antibodies recognizing β-actin (Sigma-Aldrich, St Louis, MO, USA), type IV collagen, laminin, β-catenin, Wnt3α, versican, alkaline phosphatase (ALP), p-GSK3β, GSK3β, p-ERK, ERK, p-AKT, and AKT (Abcam, Cambridge, MA, USA). Protein detection was performed with Lumi Femto (Daeil Laboratory Service, Seoul, Korea) and the Molecular Imager ChemiDoc XRS+ station. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyze and quantify the Western blot image.
Band images were obtained by using the ECL system (Thermo Fisher Scientific, Waltham, MA, USA) and Molecular Imager ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA). ImageJ software was used for the quantitative analysis of Western blot band images.
Band images were obtained by using the ECL system (Thermo Fisher Scientific, Waltham, MA, USA) and Molecular Imager ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA). ImageJ software was used for the quantitative analysis of Western blot band images.
4
Protein Expression Analysis in Kidney Tissue
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Kidney tissue lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham). After incubation with primary antibodies including type IV collagen (1:5,000) (Abcam), fibronectin (1:5,000) (Abcam), α-tubulin (1:10,000) (Sigma-Aldrich), p-p38 (1:500) (Cell Signaling), p38 (1:500) (Cell Signaling), ERK1/2 (1:500) (Cell Signaling), p-ERK1/2 (1:500) (Cell Signaling), p-Smad3 (1:500) (Cell Signaling), and p-AMPK (Cell Signaling) at 4°C overnight, the membranes were incubated with HRP-conjugated secondary antibody (1:5,000) (Amersham, Little Chalfont, United Kingdom) for 1 h. The bands were visualized with ECL and analyzed quantitatively by densitometry using LAS-4000 Imaging System (FUJIFILM, Japan).
5
Immunofluorescence Staining of Kidney Cells
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Histopathologic slides and cultured HK‐2 cells were incubated with primary antibodies FSHR (1:100; Abcam), type IV collagen (1:100; Abcam), AQP1 (1:100; Abcam), and secondary antibodies including Alexa Fluor 488 and Alexa Fluor 594. Nuclei were counterstained with 4′‐6‐diamidino‐2‐phenylindole (DAPI). Images were captured using a Nikon microscope.
6
Immunostaining of Myofiber Constructs
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Cells encapsulated in the Fib/Mtr gel were fixed with 2% paraformaldehyde in PBS overnight at 4 °C. Following fixation, samples were washed with phosphate buffered saline (PBS) and then incubated in blocking solution (5% bovine serum albumin with 0.2% Triton-X 100) (Sigma-Aldrich) for 12 h30 (link),31 (link). The myofiber sheet with Fib/Mtr gel was treated with primary antibodies (sarcomeric α-actinin (1:500) (Abcam, Cambridge, MA, USA), laminin (1:500) (Abcam), type IV collagen (1:500) (Abcam), myosin heavy chain (1:200) (R&D Systems, Minneapolis, MN, USA), myogenin (1:500) (Thermo Fisher Scientific)) or AlexaFluor 568-conjugated phalloidin at 4 °C overnight. After washing with PBS, the tissue constructs were treated with fluorescently labeled secondary antibodies (1:800) (Thermo Fisher Scientific) for 2 h at 37 °C. For nuclei staining, tissue constructs were incubated with Hoechst33258 (Dojindo Laboratories, Kumamoto, Japan) for 5 min at RT. Images were acquired using a Zeiss 510 inverted confocal microscope and analyzed using LSM Image Software.
7
SDS-PAGE Detection of Collagen and Fibronectin
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Whole cell extracts were loaded and separated by SDS-PAGE under reducing condition, as previously described. The proteins were detected by antibodies against type IV collagen (Abcam) and fibronetic (Santa Cruz Biotechnology, CA, USA), and were visualized using a horseradish peroxidase-linked goat anti-rabbit IgG (Santa Cruz Biotechnology), which was followed by enhanced chemiluminescence (Amersham, Arlington Heights, IL, USA).
8
Analyzing Kidney Collagen and Smad Signaling
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Isolated whole kidneys were lysed in radioimmunoprecipitation (RIPA) buffer and subjected to Western blot analysis. Immunoblots were probed with type IV collagen (#6586; Abcam) and phosphorylated Smad1/5/8 (#95115; Cell Signaling Technology) antibodies followed by their respective HRP-conjugated secondary antibodies. γ-Tubulin (sc-7396; Santa Cruz Biotechnologies, Inc.) was used as loading control. SuperSignal WestPico chemiluminescence substrate (Thermo Scientific Inc.) was used for visualizing the blots.
9
FACS-Isolated Fibroblast Characterization
10
Kidney Protein Extraction and Analysis
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The proteins of the kidney tissues were extracted using a Pro-Prep Protein Extraction Kit (Intron Biotechnology, Inc., Seongnam, Korea) according to the manufacturer’s instructions. The protein concentration was measured using a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). Equal kidney protein samples were separated by 12 or 15% SDS-PAGE and transferred to nitrocellulose membranes. For immunodetection, the blots were incubated overnight at 4°C in PBS containing 0.1% Tween-20 and 5% skim milk with primary antibodies raised against the following proteins: AT1R (Santa Cruz Biotechnology, Santa Cruz, CA), Copper/zinc superoxide dismutase (Cu/ZnSOD) (Enzo Life Sciences, Inc., NY), Manganese-superoxide dismutase (MnSOD), catalase, type IV collagen, and SGLT2 (all from Abcam); β-actin (Sigma-Aldrich); The second antibody was HRP-linked anti-Rabbit IgG (Cell Signaling Technology, Beverly, MA) for AT1R, Cu/ZnSOD, SGLT2, and HPR-linked anti-Mouse IgG (Cell Signaling Technology) for MnSOD and β-actin. The protein bands were detected using a chemiluminescence imaging Systems (Fusion SL4-3500, Viber-Lourmat, France), and band densities were measured by Quantity One software (Bio-Rad).
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