Cells were grown on 24-well plates during 24 h before hydrostatic pressure treatment (40 mmHg, 24 h). Then, they were fixed with a paraformaldehyde solution (4%) before incubation with anti-laminin antibody (1:400, Abcam, UK) overnight at 4°C. After extensive washing, plates were incubated for 2 h at room temperature with green-fluorescent donkey anti-rabbit IgG (H+L) secondary antibody (1:500, Invitrogen, USA). 4',6-diamidino-2-phenylindole was used to stain the cell nuclei (blue). Finally, cells were observed under a fluorescence microscope (IX71, Olympus, Tokyo, Japan) and images were analyzed with Image-Pro Plus software (Image-Pro Plus 6.0, Media Cybernetics, Inc., Silver Spring, MD, USA).
Anti laminin antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-laminin antibody is a laboratory reagent used to detect and localize laminin, a major component of the extracellular matrix. It can be used in various techniques, such as immunohistochemistry, immunocytochemistry, and Western blotting, to study the distribution and expression of laminin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscopy, by Zeiss (3 mentions)
Donkey anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Alexa fluor 488 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Dm2500, by Leica (1 mentions)
Fv1000d, by Olympus (1 mentions)
Cell sense digital imaging system, by Olympus (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Tcs sp5 confocal laser scanning microscope, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Transwell insert, by Corning (1 mentions)
12 protocols using anti laminin antibody
1
Laminin Expression Under Hydrostatic Pressure
2
Muscle Fiber Cross-Sectional Analysis
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The 14-day muscle tissue samples were used for morphological analysis. Laminin staining was performed to determine the muscle fiber cross-sectional area (CSA), as previously described (18 (link), 22 (link)). Briefly, the TA muscle samples were obtained, fixed, and cut into 10-μm thick cryo-sections. Next, the sections were incubated with an anti-laminin antibody (Abcam plc., Cambridge, UK) at 4°C for 12 hours and afterward with a fluorescent secondary antibody (Invitrogen Alexa Fluor, Thermo-Fisher Scientific, Waltham, MA, USA) at room temperature for one hour. The sections were investigated and photographed under fluorescence microscopy (Zeiss Microscopy, Jena, Germany), and the muscle fiber CSA was determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Quantifying Muscle Fiber Cross-Sectional Area
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The fiber CSA of TA muscles was detected using laminin staining. Briefly, mouseTA muscles were fixed in 4% paraformaldehyde at room temperature. Subsequently, TA muscles were flash frozen in embedding medium and sectioned on cryostat with 10-μm thickness. The cryosections were placed on glass slides. After blocking and washing, the slides were incubated for 12 h at 4°C with anti-laminin antibody (1:200; Abcam, Cambridge, UK). Sections were subsequently incubated with the Alexa Fluor secondary antibody (1:400; Invitrogen Antibodies, Waltham, MA, USA) for 30 min at room temperature. Then, the slides were imaged by fluorescence microscopy (Zeiss, Germany), and the CSA of myofibers were determined through a blinded analysis with the ImageJ software (NIH, Bethesda, MD, USA) of five randomly captured muscle images from each experimental condition.
4
Histological Analysis of Mouse Tissues
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Harvested mouse tissues from the liver, adipose tissue, or skeletal muscle were fixed in 10% formalin and then mounted in OCT for cryosectioning or paraffin for immunohistochemical staining. The frozen samples were cryosectioned at a thickness of 10 μm. Sections were then blocked in 1% BSA in PBS for 1 h and incubated with an anti-laminin antibody (Abcam, Cambridge, MA, USA) overnight at 4 °C. The Alexa Fluor 488 (Abcam, Cambridge, MA, USA) fluorescent dye conjugated to a secondary antibody and DAPI (Vector Laboratories, Burlingame, CA) were used for visualization. The paraffin sections were stained with hematoxylin for 30 s and then with eosin for 60 s. Measurements were performed using NIH ImageJ software (http://rsb.info.nih.gov/ij ).
5
Muscle Fiber Cross-Sectional Area Analysis
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The muscle fiber CSA was measured using laminin staining as previously described (4 (link),6 (link)). Briefly, all mouse tibialis anterior muscles were fixed, flash-frozen, and cryosectioned into 10-µm thick sections. The middle third of each muscle was sliced. The sections were incubated in the presence of anti-laminin antibody (Abcam) at 4 °C for 12 h, and then incubated with fluorescent secondary antibody (Alexa Fluor, Invitrogen, USA) at room temperature for 1 h. Five slices were selected from each muscle sample. The slides were observed and imaged under fluorescence microscopy (ZEISS, Oberkochen, Germany) and the CSA was determined using ImageJ software [National Institutes of Health (NIH), Bethesda, MD, USA] through blinded analysis of five randomly captured images under each detection condition.
6
Histological Analysis of Skeletal Muscle
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The histological analysis was performed as described previously (Zhang et al. 2019 (link)). The cross-sectional area and minimal Feret’s diameter of the gastrocnemius muscles were determined by immunostaining with an anti-laminin antibody (dilution: 1:200; Abcam, Cambridge, MA, USA) (Moresi et al. 2010 (link)). The primary antibody was detected by Alexa Fluor-594 (dilution: 1:1000; Life Technologies, Carlsbad, CA, USA) fluorescent dye conjugated to an anti-rabbit secondary antibody. Myotubes were fixed with 4% paraformaldehyde for 10 min, blocked with 5% goat serum for 30 min, and incubated with anti-myosin (dilution: 1:500; ZSGB-BIO, China) at 4 °C overnight. The secondary antibody was goat anti-mouse IgG H&L (Alexa Fluor® 488) (dilution: 1:1000; Life Technologies, Carlsbad, CA, USA). Nuclei were stained using Vectashield with DAPI (Vector, USA). Images were visualized through a Leica TCS SPS III confocal microscope (Leica, Germany). The diameter of each individual myotube was measured and the average overall diameter was calculated. Images were analyzed using Image-Pro Plus 6.0.
7
Muscle Fiber Composition Analysis
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Immunohistochemistry was carried out using anti‐myosin heavy chain type 2A and 2B antibodies (DSHB, IA, USA) and an anti‐laminin antibody (Abcam, Cambridge, UK). Counterstaining was conducted using 1 μM DAPI solution. Muscle fibre CSA was measured with the ImageJ 1.48 software (National Institutes of Health, USA) after the images were visualized with fluorescence microscopy (LEICA DM 2500).
8
Laminin-based Muscle Fiber CSA Analysis
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The fiber CSA of TA muscles was detected by using laminin staining. Briefly, rat TA muscles were fixed in 4% paraformaldehyde at room temperature. Subsequently, TA muscles were flash-frozen in embedding medium, and sectioned on cryostat with 10-μm thickness. The cryosections were placed on glass slides. After blocking and washing, the slides were incubated for 12 h at 4°C with anti-laminin antibody (1:200; Abcam, Cambridge, United Kingdom). Sections were subsequently incubated with the Alexa Fluor secondary antibody (1:400; Invitrogen Antibodies, Waltham, MA, United States) for 30 min at room temperature. After washing, CSAs of myofibers were determined through a blinded analysis with the ImageJ software (NIH, Bethesda, MD, United States) of five randomly captured muscle images from each experimental condition.
9
Matrigel Coating of Gelatin Microspheres
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The matrigel solution (20 μl) was dropped onto 2 mg of freeze-dried gelatin hydrogel microspheres (GM), followed by leaving at 4 °C for overnight to allow matrigel to absorb onto the microspheres. The matrigel solution was completely absorbed into the GM because the solution volume was much less than that theoretically required for the equilibrated swelling of microspheres. To evaluate the matrigel existence on the surface of matrigel-coated GM were incubated with an anti-laminin antibody (Abcam Inc., Cambridge, UK) for 60 min at 25 °C and subsequently Alexa Fluor® 488 Donkey anti-rabbit (Thermo Fisher Inc., Massachusetts, America) for 30 min at 25 °C, followed by fluorescent viewing with confocal laser scanning microscope (FV1000D, Olympus Ltd, Tokyo, Japan).
10
Characterizing Muscle Fiber Composition
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Formalin-fixed paraffin-embedded sections of mouse muscle (5um thickness) from gastrocnemius and quadriceps were cut with a microtome and mounted on charged slides. Sections were either subjected to standard hematoxylin-eosin (H&E) staining for overview, or immunolabeled with the following antimyosin heavy chain (MyHC) isoform antibodies: type I MyHC isoform (Abcam, Cat# ab11083); type II MyHC isoform (Abcam, Cat# ab51263). The sections were co-stained with an anti-laminin antibody (Abcam, Cat# ab11575) to allow measurement of fiber size. In all protocols, donkey anti rabbit Alexa-488 conjugated secondary antibody was used for laminin staining, donkey anti-mouse Alexa-594 conjugated secondary antibody for type I MyHC antigen staining, and anti-mouse Alexa-488 conjugated secondary antibody for type II MyHC antigen staining. Photomicrographs were acquired using Olympus BX 51 and IX 71 microscopes equipped with Cell Sense digital imaging system (Olympus, Japan). To assess the cross-sectional area (CSA) of the different myofiber types, digitized photographs were acquired and the myofiber CSA was automatically measured by means of ImageJ 1.45 g (NIH, freeware imaging software).
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