Rabbit anti laminin antibody

After the antigen retrieval step using 1 mmol/L EDTA (pH 8.0), 4-μm-thick paraffin sections were incubated with the primary antibody for laminin and CD31 staining. Rabbit anti-laminin antibody (1:30 dilution; Sigma-Aldrich, St. Louis, MO, USA) and mouse anti-CD31 antibody (1:100 dilution; CST, Boston, USA) were diluted in blocking serum at 4°C overnight, washed with PBS three times, and then treated with Alexa 488-conjugated goat anti-rabbit antibody or Alexa 647-conjugated goad anti-mouse antibody (1:500 dilution; Abcam, Cambridge, MA, USA). Then, all sections were followed by DAPI (Invitrogen, Carlsbad, CA, USA) for nuclear staining. All immunofluorescence images were captured by a fluorescence microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan) and NIS-Elements D 4.50 (Nikon Instruments, Tokyo, Japan). The numbers of positive cells were counted in three randomly selected images from grafts were averaged and are presented as means ± SD. The blood vessels quantification was analyzed with Image J software practicing the “analyze particle”.

For histology, the TA and diaphragm muscles were harvested, embedded in OCT compound (Leica Biosystems), and then frozen in isopentane cooled in liquid nitrogen. Serial 10 μm cryosections were collected. For quantification of myofiber size and numbers, sections were stained for laminin. Muscle sections were fixed in 4 % PFA for 30 min, washed three times with PBS, blocked with 5 % BSA in PBS for 1 h at room temperature (RT), and then incubated with rabbit anti-laminin antibody (1:400; Sigma) overnight at 4 °C overnight. The following day, slides were washed three times in PBS and then stained with goat Alexa-555 anti-rabbit secondary antibody (Invitrogen) for 45 min at RT. To stain nuclei, we used ProLong^® Gold Antifade reagent along with DAPI (molecular probes). Hematoxylin and eosin (H&E) staining was used for assessment of centrally located nuclei myofibers. Masson’s trichrome staining was utilized to detect collagen content (Polysciences, Inc.). Fiji ImageJ software was used to quantify myofiber size and numbers, as well as muscle cross-sectional area. To quantify the collagen, the colors from the figure were split and a threshold was used to identify the blue staining. Then, the complete muscle section was selected as a region of interest, and the percentage of area depicted by the threshold (equivalent to the collagen area) was measured also using ImageJ software.

Antibodies and reagents were obtained from the following sources. PE-conjugated anti-CD31, anti-CD45, and anti-Sca-1 and APC-conjugated anti-Vcam1 antibodies were obtained from BioLegend (San Diego, CA, USA). Rabbit or mouse anti-GFP antibodies cross-reacting with YFP were obtained from Thermo Fisher Scientific (Carlsbad, CA, USA) or EMD Millipore. Mouse anti-PAX7 and mouse anti-myosin heavy chain (MF20, MAB4470) antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Rabbit anti-MyoD antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-Laminin antibody was obtained from Sigma (Sigma-Aldrich, St. Louis, MO). Rat anti-Laminin α2 antibody was obtained from Enzo (Enzo Life Sciences, NY). Rabbit anti-Dystrophin antibody was obtained from Abcam (Cambridge, MA, USA). Rat anti-Ki67 antibody and DAKO Protein Block were obtained from DAKO (Tokyo, Japan). Alexa Fluor-conjugated secondary antibodies were purchased from Thermo Fisher Scientific. M.O.M. kit and mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) for nuclear staining was obtained from Vector Laboratories (Burlingame, CA, USA).

Transverse serial sections of FDP muscle (10 μm thick) were obtained using a cryostat maintained at − 25 °C (HM-560, Microm H, Thermo Scientific, Waltham, Massassuchets, USA). Sections were stored at − 20 °C until histochemical staining. Before labeling, the sections were dried and fixed for 10 min in acetone. The sections were then washed in PBS, blocked and permeabilized with PBS 0.1% Triton X-100 and 20% horse serum.
For cross-sectional area (CSA) determination, the sections were incubated for 1 h with a rabbit anti-laminin antibody (1/400) (Sigma Aldrich, Saint-Louis, Missouri, USA), washed and then incubated with a secondary antibody conjugated to ALEXA 488 (goat anti-rabbit, Sigma, 1:800).
For muscle fiber typing determination, the sections were incubated with anti-MHC primary antibodies (anti-slow (I) MyHC, BA-D5, Developmental Studies Hybridoma Bank, 1:10 and anti-fast (II) MyHC, M4276, Sigma-Aldrich, 1:200) for 1 h at 37 °C, followed by washes in PBS and incubation with the secondary antibodies (1/800) (ALEXA 488; ALEXA 568, A11031, Invitrogen, Carlsbad, California, USA,) for 1 h.
The sections were scanned using a Nanozoomer (Hamamatsu), and CSA and fiber typing and were determined using ImageJ® software (version 1.46r).