Experimental methods and CSA analysis were based on previous studies42 (link)–44 (link). The GAS and TA muscles were cut into 10-μm-thick frozen sections using a cryostat (CM-502, Sakura Finetech). Samples were blocked with 5% goat serum (PCN5000, Thermo Fisher Scientific Japan, Tokyo, Japan) for 1 h, incubated with a primary anti-laminin antibody (L8271, 1:1000, Sigma Aldrich Japan, Tokyo, Japan) for 2 h. After incubation, the cells were washed with 0.1 M phosphate buffer (5 min × 3 times) and incubated with Alexa fluor 488 conjugated secondary antibody (1:2000, A-11008, Thermo Fisher Scientific, MA, USA) for 2 h at room temperature. Images were captured using a confocal laser microscope (FV-3000; Olympus, Tokyo, Japan) and quantified using MyoVision (University of Kentucky). Twelve images were acquired from four sections per muscle for analysis using approximately 4500 to 8000 fibers per group.
L8271
Manufactured by Merck Group
Sourced in United States, Japan
The L8271 is a laboratory equipment product from Merck Group. It is a high-performance device designed for specific laboratory applications. The core function of the L8271 is to perform precise measurements and analyses as required in scientific research and testing environments.
Automatically generated - may contain errors
Lab products found in correlation
Pcn5000, by Thermo Fisher Scientific (2 mentions)
A11008, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Fv3000, by Olympus (2 mentions)
Ab90395, by Abcam (2 mentions)
Ab45688, by Abcam (2 mentions)
Ab6311, by Abcam (2 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (2 mentions)
Vectastain universal elite abc kit, by Vector Laboratories (2 mentions)
Laminin, by Merck Group (1 mentions)
Collagen type 4, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Collagen type 1, by Santa Cruz Biotechnology (1 mentions)
L6274, by Merck Group (1 mentions)
Dm750, by Leica (1 mentions)
P10144, by Thermo Fisher Scientific (1 mentions)
Sl038, by Solarbio (1 mentions)
Cm1900, by Leica (1 mentions)
G1120, by Solarbio (1 mentions)
5 protocols using l8271
1
Muscle Fiber Analysis via Immunofluorescence
2
Laminin-based Muscle Fiber Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experimental methods and CSA analysis were modified from those in previous studies38 (link)–40 (link). GAS and TA muscle bellies were cut into 10-μm-thick frozen sections using a cryostat (CM-502, Sakura Finetech Japan).
Samples were blocked with 5% goat serum (PCN5000, Thermo Fisher Scientific Japan, Tokyo, Japan) for 1 h and incubated with primary anti-laminin antibody (L8271, 1:1000, Sigma Aldrich Japan, Tokyo, Japan) for 2 h. The primary antibody was diluted with a blocking reagent at room temperature. After incubation, the samples were washed with 0.1 M phosphate buffer (5 min × 3 times) and incubated with Alexa fluor 488 conjugated secondary antibody (1:2000, A-11008, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 2 h. After incubation, samples were washed with 0.1 M phosphate buffer (5 min × 3 times) and mounted with fluorescence anti-fading reagent (12593-64, Nacalai Tesque, Kyoto, Japan). Images were captured using a confocal laser microscope (FV-3000; Olympus, Tokyo, Japan) and quantified using MyoVision (University of Kentucky, Kentucky, USA). Twelve images were acquired from four sections per muscle for analysis using approximately 4500 to 8000 fibers per group.
Samples were blocked with 5% goat serum (PCN5000, Thermo Fisher Scientific Japan, Tokyo, Japan) for 1 h and incubated with primary anti-laminin antibody (L8271, 1:1000, Sigma Aldrich Japan, Tokyo, Japan) for 2 h. The primary antibody was diluted with a blocking reagent at room temperature. After incubation, the samples were washed with 0.1 M phosphate buffer (5 min × 3 times) and incubated with Alexa fluor 488 conjugated secondary antibody (1:2000, A-11008, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 2 h. After incubation, samples were washed with 0.1 M phosphate buffer (5 min × 3 times) and mounted with fluorescence anti-fading reagent (12593-64, Nacalai Tesque, Kyoto, Japan). Images were captured using a confocal laser microscope (FV-3000; Olympus, Tokyo, Japan) and quantified using MyoVision (University of Kentucky, Kentucky, USA). Twelve images were acquired from four sections per muscle for analysis using approximately 4500 to 8000 fibers per group.
3
Protein Expression Analysis via Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One drop of each sample of soluble protein was placed in a nitrocellulose membrane. After drying, membranes were incubated for 1 h with 5% BSA, with agitation, at room temperature. Subsequently, membranes were incubated with primary antibody:mouse antiCollagen type I 1:1000 (#ab90395, abcam, Cambridge, UK); rabbit antiCollagen type IV, 1:500 (#ab6311, abcam, Cambridge, UK); rabbit antifibronectin 1:500 (#ab45688, abcam, Cambridge, UK); and mouse antilaminin, 1:500 (#L8271, Sigma-Aldrich, St. Louis, MO, USA). After overnight incubation, membranes were washed 3× for 5 min with Tris Buffered Saline (TBS) with Tween 20 and then the R.T.U. VECTASTAIN® Universal ABC Elite® Kit (#PK-7200, Vector Laboratories, Burlingame, CA, USA) was used as a secondary antibody, in accordance with manufacturer’s instructions. Finally, incubation was revealed using a Peroxidase Substrate Kit (DAB) (#SK-4100, Vector Laboratories, Burlingame, CA, USA). Extraction buffer without samples was used as a negative control. Collagen type I (#sc-136157, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Collagen type IV (#C5533, Sigma-Aldrich), fibronectin (#FC010, Sigma-Aldrich, St. Louis, MO, USA) and laminin (#L6274, Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. At least three independent samples were used in each condition.
4
Immunolocalization of Extracellular Matrix Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunolocalization of different proteins, such as collagen type I, collagen type IV, fibronectin and laminin, was performed in paraffin-embedded samples sectioned at 5 μm, as previously described for collagen type II [30 (link)]. Briefly, samples were incubated with mouse anticollagen type I 1:100 (#ab90395, abcam, Cambridge, UK); rabbit anticollagen type IV, 1:50 (#ab6311, abcam, Cambridge, UK); rabbit antifibronectin 1:300 (#ab45688, abcam, Cambridge, UK); and mouse antilaminin, 1:300 (#L8271, Sigma-Aldrich, St. Louis, MO, USA), overnight at 4 °C in a humidified atmosphere. As a secondary antibody, the R.T.U. VECTASTAIN® Universal ABC Elite® Kit (#PK-7200, Vector Laboratories, Burlingame, CA, USA) was used, in accordance with manufacturer’s instructions. Incubation was revealed using a Peroxidase Substrate Kit (DAB) (#SK-4100, Vector Laboratories, Burlingame, CA, USA). Samples were counterstained with hematoxylin and mounted in an aqueous mounting medium. Slides were observed in an optical microscope with a coupled camera (DM750, Leica, Wetzlar, DE, Germany). At least three independent samples were used in each condition.
5
Microscopic Evaluation of Nerve Scaffold Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The two obtained kinds of ANXs (CD NG, CD + scCO2 NG) were embedded in OCT embedding glue (SAKURA, Japan), and then frozen sections with a thickness of 6 μm were made using a freezing microtome (CM1900, Leica, USA) on the cross-section of the nerve. At the same time, natural porcine sciatic nerve (Natural SN) was selected as a control. After putting in PBS and degumming, hematoxylin and eosin (H&E) staining was performed according to the kit instructions (G1120, Solarbio, China). The tissue on the section was blocked in 10% goat serum (SL038, Solarbio, China) for 30 min. Laminin antibody (1:1000, L8271, Sigma–Aldrich, USA) from mice was used as the primary antibody to cover the nerve tissue and incubated overnight in the dark at 4 °C. The excess primary antibody was washed off with PBS and then incubated for 2 h with goat anti-mouse IgG H&L secondary antibody (Alexa Fluor 594, 1:200, ab150116, Abcam, USA) in the dark at 37 °C. The excess secondary antibody was washed off with PBS, and sections were incubated with DAPI (1:100, 62248, Thermo Fisher Scientific, USA) staining solution for 5 min in the dark, washed with PBS and mounted with aqueous mount (P10144, Thermo Fisher Scientific, USA). Finally, a microscope equipped with a DP71 camera (BX51, Olympus, Japan) was used to image the stained sections obtained in the above experiment.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!