Bx41 bright field microscope

To determine the extent of myelination, every 4^th section (PND14) or every 6^th section (PND270) was stained for myelin basic protein (MBP) to assess global development and developmental trajectory of myelination in the CC (separately examining truncus (immediately posterior to the genu), medial external capsule and lateral external capsule), striatum, and frontal cortex. Briefly, brain sections were washed of cryoprotectant and placed into primary antibody for MBP (EMD Millipore; MAB386). Tissue was then placed into biotinylated secondary antibody (Vector labs; BA-9401; 1:200 dilution) for 1 hour and the stain was visualized using 3-3′-diaminobenzidine (DAB). Immunolabeled tissue was mounted onto Superfrost Plus micro slides (VWR; Radnor, PA; 48311-703) and cover-slipped using Cytoseal 60 (Fisher Scientific; Pittsburgh, PA; 23-244257).
Briefly, at least 3 images per region of interest were captured using an Olympus BX-41 brightfield microscope prior to image analysis. Expression of MBP was analyzed using Image Pro Plus 7.0 (Mediacybernetics) using segmentation and thresholding to estimate a percent area of the region of interest that was positive for MBP expression. For CC, percent of area myelinated was determined, whereas total relative units of myelination were measured in frontal cortex and striatum.

An Olympus Fluoview FV 300 confocal system featuring three lasers and an Olympus BX60 fluorescent microscope (Olympus America, Melville, NY) was used to collect images of NMJs. Using a 100x oil immersion objective, it was initially established that the entire NMJ was within the longitudinal borders of the myofiber and that damage to the structure had not occurred during sectioning. A detailed image of the entire NMJ was constructed from a z-series of scans taken at 1 µm thick increments. Digitized, two-dimensional images of NMJs were stored on the system’s hard drive and later quantified with the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). A minimum of 10 NMJs per muscle were quantified and measurements were averaged to represent synaptic morphology within that muscle.
In the quantification of muscle fiber profiles, an Olympus BX41 bright field microscope was used in conjunction with a 40X objective. Myofiber cross-sectional areas were quantified with the Image Pro-Express software. A random sample of 150–200 myofibers from each muscle was analyzed to determine average myofiber size (i.e., cross-sectional area) and fiber type composition (% of total number of fibers examined) for that muscle.