Mbf microscope

Two sites along the musculocutaneous nerve (distal to lateral cord and 2 mm proximal to biceps) were fixed overnight with a mixture of 2% PFA and 2.5% glutaraldehyde in 0.1 M PB, followed with overnight fixation using 1% osmium tetroxide. The tissue was then dehydrated in graded ethanol (30%, 50%, 70%, 80%, 90%, 95% for 5 minutes each and 100% for 3 times in 30 minutes) and infiltrated with propylene oxide (PO) twice in 30 minutes, PO: Epon (1:1) for one hour and pure Epon overnight. After that, the nerve was embedded in Epon and polymerized at 60 °C for 72 hours. Semithin sections (0.5 μm) were cut by a microtome (Ultracut) using a glass knife before staining with 0.5% toluidine blue in 1% borax for 35 s. A light microscope (MBF Nikon Microscope) was used for observation and images were digitalized. Around 80 axons from each animal were randomly selected for area measurement using image J. Student’s t test was applied for statistical analysis. Ultrathin sections of 90 nm thickness were stained with 3% uranyl acetate and 1% lead citrate and digital images captured by electron microscopy (TEM, Phillip model 208).

Cross and longitudinal sections of biceps were cut and processed for H&E staining. Briefly, sections were deparaffinized by toluene and rehydrated by degraded ethanol (100%, 100%, 95%, 95%, 75% ethanol, 2 minutes each). Nuclei were stained with Harris haematoxylin for 8 minutes, followed by differentiation with 0.3% acid alcohol. Cytoplasm was stained with eosin for 2 minutes. Sections were then subject to dehydration by graded ethanol (75%, 95%, 95% and 100%, 2 dips each; 100% for 2 minutes; 100% for 12 minutes). Light microscopy (MBF Nikon Microscope) was used for observation and images were digitalized. Around 200 muscle fibers from each biceps were randomly selected for diameter assessment. Z test was applied for statistical analysis. 10 photos were randomly digitized from each biceps and fibroblast nuclei on each photo were quantified. The ratio of the number of fibroblast nuclei in the ipsilateral biceps to that on the contralateral side was computed. The data was expressed as mean ± SEM with a student’s t test as statistical analysis.

Fixed ipsilateral and contralateral biceps (from 12-week time point rats) were sectioned longitudinally from the ventromedial to superficial side, at a thickness of 14 μm. Every 4th section was collected and 20 slides for each muscle were harvested to stain with α-Bungarotoxin (α-BTX, Invitrogen, Alexa Fluor 594 conjugated, 1:500) for 30 minutes. Numbers of motor endplates (MEP) on each section were counted. 10–15 MEPs from each section and 150–200 MEPs from one muscle were randomly chosen to take photos (MBF Nikon Microscope). MEP area was measured using image J. Area range was divided into six categories: from 0 to 500 μm² (separated by hundreds) and over 500 μm². For every biceps, numbers of MEPs falling into each category were counted and the proportion to the total number calculated. Data was expressed as mean ± SEM. One-way ANOVA was used for MEP number assessment, while student’s t test was applied for MEP area distribution analysis.