Bx51 light microscope

After behavioral tests, six mice in each group were anesthetized with sodium pentobarbital (50 mg/kg) and perfused intracardially with 0.9% saline followed by 4% paraformaldehyde (PFA) in saline. Serial coronal sections (30 μm) of the brain were made on a freezing microtome and then processed for immunohistochemistry using an anti-myelin basic protein (MBP) rabbit polyclonal antibody (1:200 dilution, Sigma Aldrich, St. Louis, MO, USA). Images were obtained using a Nikon BX-51 light microscope.

Heart and kidney were removed after euthanasia. Fresh tissue was dissected, cleaned with ice-cold saline and fixed at 4 °C with 4% paraformaldehyde for 12 h. Fixed tissues were dehydrated by graded ethanol immersion and cleared by xylene. Tissues were then embedded in paraffin and thereafter, sliced into 3-µm-thick sections. Sections from kidney and heart were stained with hematoxylin and eosin (H&E) for structural inspection. Histological analyses were examined under Olympus BX51 light microscope (Tokyo, Japan) and NIS-Elements BR Analysis 4.00 (Nikon Instruments Inc., NY, USA). Measurement of cardiac myocyte diameter was performed as previously described²² . The diameters were obtained by measuring across the center of each cardiac myocyte at 100× magnification. The myocyte must be longitudinally cut with the presence of nuclei. For Bowman’s capsule measurement was modified from the method of Lindahl and co-workers^{23 (link)}. Fifty Bowman’s capsules pictures (20× magnification) per animal were used for analysis. Total Bowman’s capsule area and glomerular tuft area were derived from tracing the outline of parietal layer and visceral epithelial layers, respectively. The difference value between total Bowman’s capsule area and glomerular tuft area was defined as Bowman’s space area.