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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Selection of Ultrastructural Neuroplasticity Parameters for Quantitative Analysis
- Mapping the area of interest
- Choose an area of interest (e.g., hippocampus or cerebellum) and localize the area in the section atlas by choosing the image from the atlas that contains this area.
- Sketch the borders of the area of interest onto the section image and find/superimpose these region borders onto the resin specimen.
- Scratch-mark the borders of the area of interest (e.g., hippocampus or cerebellum) on the resin specimen, using a fine needle gauge (26 G, 1 in).
- Heat the resin specimen to 85 °C in an oven to soften the resin for trimming or, alternatively, use a trimming device, a thin blade, or sandpaper.
- Excise the area of interest from the resin specimen with a razor blade. Mount the specimen on holding bars of acrylic glass of the required caliber (e.g., with a diameter of 8 mm and a length of 1 cm) with glue. Trim the mounted specimen for semi- and ultrathin sectioning.
- Prepare semithin (0.75 µm) and ultrathin (70 nm) sections of the trimmed area using an ultramicrotome: set it at 1.5 mm/s for 0.75 µm thickness and at 0.7 mm/s for 70 nm thickness.
- Collect the semithin sections on glass carriers and stain the sections with 1% toluidine blue in PBS (for 4 min).
- Wash the sections several times in deionized water. Examine the stained sections under the light microscope using 4x (NA of 0.1 ∞/-), 10x (NA of 0.22 ∞/0.17), 40x (NA of 0.65 ∞/0.17), and 100x (NA of 1.25 ∞/0.17) objectives.
- Collect ultrathin sections on nickel grids. Subject the grids to TEM at 180 kV and at 3,200x, 6,000x, and/or 8,000x magnification.
- TEM Analysis
- Choose the ultrastructural parameters of interest for quantitative TEM analysis (e.g., boutons with vesicles and mitochondria or myelinated and nonmyelinated axons) and take TEM images of these parameters under 3,500x, 6,000x and/or 8,000x magnification.