Dm4000b compound microscope

After GMI was assessed, the gastrocnemius muscles were fixed in formalin. Routine H&E-stained paraffin sections were prepared after the cross-sections of the muscle samples were taken. Six non-overlapping fields were chosen from each muscle sample, with three hundred muscle fibers assessed in total. Images were taken using a Leica DM4000B Compound Microscope (Leica Microsystems, Germany) with a Qimaging Retiga 2000R Color Digital Camera (Teledyne Photometrics, USA), then digitized and assessed using Image-Pro Plus, Ver 6.3.0.512 (Media Cybernetics, USA). The average muscle fiber areas were compared between the right and left limb in each animal, and the values were expressed as the R/L ratio.

Expression of growth factors involved in nerve regeneration was assessed by monoclonal antibodies and immunofluorescence techniques. Freshly dissected nerve conduit from both the proximal and distal stump was suspended and snap-frozen in O.C.T. compound. Tissue slides were cut for 1 μm slides and fixed for 10 min in acetone. Next, the sections were rinsed in Tris Buffered Saline (TBS, Agilent Technologies, Inc., USA) and incubated with mouse antirat vWF, VEGF (Thermo Fischer Scientific, USA) and S-100 (Abcam, Inc., UK), rabbit antirat GFAP (Thermo Fisher Scientific, USA), Laminin B and NGF (Abcam, Inc., UK), and mouse anti-human HLA-1 and HLA-DR (Abcam, Inc., UK) monoclonal antibody for 30 min. Incubation using secondary antibodies was performed using goat anti-mouse or goat anti-rabbit IgG Cross-Absorbed Alexa Fluor 488 (Thermo Fischer Scientific, USA). PKH26 staining of hMSC prior to implantation assessed the presence of hMSC in the conduit. The slides were stained with DAPI and analyzed using a Leica DM 4000B Compound Microscope (Leica Microsystems, Germany) with a Qimaging Retiga 2000R Color Digital Camera (Teledyne Photometrics, USA) and digitalized and assessed using Image-Pro Plus, Ver 6.3.0.512 (Media Cybernetics, USA). Assessment of immunoreactivity was scored as follows: 0 = no staining; 1 = weak; 2 = moderate; and 3 = strong.