A sample of crushed nerve (about 1 cm) was excised on days 7, 14, 21, and 28 following surgery. Samples containing 30 μg of total protein from the injured nerve were subjected to 12% SDS-PAGE, electroblotted onto PVDF membranes (Biosharp), and then blocked with 5% nonfat milk for 2 h at 20°C. The membranes with the transferred proteins were incubated with the rabbit anti-GAP-43 primary antibody (1 : 400, Beijing Biosynthesis Biotechnology Co., Ltd.), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1 : 1,000) as the secondary antibody. Chemiluminescence reaction was carried out with an ECL kit (Biosharp) for 1 min, followed by exposure to a Kodak X-Omat radiographic film. Similar procedures were carried out with an anti-β-actin antibody (1 : 1,000; Beijing ZSGB-Biotechnology Co., Ltd.). Relative expression was calculated as the ratio of the gray value of the target protein band to an internal reference band.
X omat radiographic film
Manufactured by Kodak
X-Omat radiographic film is a product designed for use in medical and industrial x-ray imaging applications. It is a photographic film that captures x-ray images, enabling the visualization and documentation of internal structures and conditions.
Automatically generated - may contain errors
2 protocols using x omat radiographic film
1
Western Blot Analysis of GAP-43 Expression in Injured Nerve
2
Western Blot Analysis of Netrin-1 and VEGF
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Samples containing 50 µg of total protein from the injured gastrocnemius muscle were subjected to electrophoresis using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were then blocked with 5% fat-free milk for 2 h at 37°C, incubated with rabbit anti-Netrin-1 and anti-VEGF primary antibodies (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (IgG, 1:2000) as the secondary antibody. The chemiluminescence reaction was carried out with an enhanced chemiluminescence (ECL) kit (Bio-Rad) for 1 min and was followed by exposure using Kodak X-Omat radiographic film. Similar procedures were carried out with an anti-β-actin antibody (1:500; Santa Cruz Biotechnology), which was used for actin blotting as the loading control.
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