Gatan 832 digital camera

The lateral corpus callosum of mice was rapidly dissected and immersed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 overnight at 4 °C. Samples were processed as previously reported^{34 (link),35 (link)}. After washing three times with the same buffer, the samples were postfixed in 1% osmium tetroxide buffered with cacodylate for 1 h at room temperature, dehydrated through a graded series of ethanol and embedded in Spurr’s resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a Hitachi H-7100 electron microscope equipped with a Gatan 832 digital camera (Gatan, 86 Inc.). Nonoverlapping EM images of the coronal-sectioned corpus callosum were analyzed to determine the g-ratio as previously reported^{36 (link)}. Images were processed with ImageJ software (https://imagej.nih.gov/ij/) with a plug-in (https://gratio.efil.de) that allowed randomly selected axons to be analyzed. The outer border (used to calculate myelin area) and inner border (used to calculate axon area) of myelin sheath were manually marked, and the g-ratio was calculated as the axonal diameter divided by the myelinated fiber diameter. A total of 300 axons from 3 different images from the control mice and 600 axons from 6 different images from the PD mice were analyzed.

Electron microscopy was performed as described.^{42 (link)} Briefly, cells in 6-well plates were fixed in 2.5% glutaraldehyde (Sigma) in 0.1 M phosphate buffer (pH 7.4) for 2 h, and then dehydrated in a graded ethanol series and embedded. Ultrathin sections were mounted on copper grids. The samples were then stained and visualized using a 120 kV Jeol electron microscope (JEM-1400, Peabody, WA, USA) at 80 kV. Images were captured using a Gatan-832 digital camera (Pleasanton, CA, USA).

Retinas were isolated and fixed in 2% glutaraldehyde and 2% PFA in 0.1 M PB at 4°C overnight. After postfixing in 1% osmium tetroxide for 1 h, tissue samples were dehydrated in a graded ethanol series and embedded in epoxy resin (EMS, Hatfield, PA, United States). Ultrathin sections (70 nm in thickness) were collected on copper grids and stained with uranyl acetate and lead citrate before examination under a Hitachi H-7100 electron microscope (Hitachi, Tokyo, Japan) equipped with a Gatan 832 digital camera (Gatan, Inc., Pleasanton, CA, United States).
For the quantification of synaptic ribbon conditions, images of the OPL were taken at a magnification of 40,000X. Approximately 500 photoreceptor terminals for each age were examined and classified into different categories of rod-shaped ribbon profiles.