Rabbit polyclonal anti laminin antibody

Calf muscles were placed in 30% sucrose PBS for 24 hours, bisected at the middle level, mounted in an OCT compound (Leica), and snap-frozen in liquid nitrogen. To determine capillary density, calf muscles were mid-bisected with one cross-section 1.5 mm above the bisecting plane and another 1.5 mm below the bisecting plane; in other words, the two sections were 3 mm apart. Both sections, fixed with methanol for 10 min and washed briefly with PBS, were labeled with a mixture of monoclonal rat anti-murine platelet-endothelial cell adhesion molecule-1 antibody (1:200; B&D Pharmingen, San Diego, CA, USA) and polyclonal rabbit anti-laminin antibody (1:100; Chemicon) at 37 °C for 2 hours, followed by incubation with a mixture of CY3-conjugated anti-rat antibody and CY5-conjugated anti-rabbit antibody (both from Chemicon). In parallel, methanol-fixed frozen sections were stained with TRITC-conjugated murine EC-specific Bandeiraea simplicifolia lectin 1 (1:50; Sigma) and polyclonal rabbit anti-laminin antibody (1:100; Chemicon) at 4 °C overnight followed by incubation with a CY5-conjugated anti-rabbit antibody. Capillaries were counted for every 30 randomly chosen high-power fields on the two sections of each animal (without an amputated hind limb). The results are expressed as capillaries per myocyte.

Soleus muscles were dissected from the mice, immediately frozen in chilled isopentane and liquid N₂, and stored at −80 °C until use. The cross-sections (10-µm thick) of soleus muscle were cut in a cryostat (CM1850; Leica, Wetzlar, Germany) at −20 °C. The muscle sections were air-dried and then fixed with 10% formalin. The sections were washed with phosphate-buffered saline (PBS) and incubated with PBS containing 10% normal serum, followed by reaction with rabbit polyclonal anti-laminin antibody (Sigma-Aldrich, St. Louis, MO, USA) diluted with PBS containing 5% normal serum and 0.3% Triton X-100. The immunoreactive sections were incubated with goat anti-rabbit immunoglobulin G (IgG) (Alexa Fluor 488 conjugate) (Life Technologies, Grand Island, NY, USA) diluted with PBS containing 5% normal serum and 0.1% Triton X-100. Immunofluorescent images were obtained with a BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan). The cross-sectional area (CSA) (µm²) of the muscle fibers was measured as described previously [25 (link)]. The mean fiber CSA was determined from more than 200 fibers per soleus muscle (n = 6–8 per group). The distribution of the CSA was plotted as frequency histograms.