Procedure: Estimation of midsagittal area of the corpus callosum

1. All histologic data for this study were obtained from The Mouse Brain Library (MBL), a physical and Internet resource containing high-resolution images of histologically processed slides from over 3200 adult mouse brains.
2. Mice are anesthetized with Avertin (0.8 mL i.p.).
3. Mice are transcardially perfused with saline followed by fixative (either a glutaraldehyde/paraformaldehyde solution or 4% paraformaldehyde).
4. Brains are removed and weighed.
5. After variable post-fixation times, brains are embedded in 12% celloidin and sliced at a width of 30 microns.
6. Area is estimated using a computer-controlled microscope and software.
7. Dorsal-ventral distance of the corpus callosum is measured on the midline of coronal sections that are spaced ~150 microns apart (9-11 sections per brain).
8. Area is computed using Cavalieri's rule to estimate the area underneath a curve described by dorsal-ventral distance on the Y-axis and rostro-caudal distance on the X-axis.
9. In cases where there are missing or damage sections, a piece-wise parabolic estimation is used.
10. Area is re-measured on 10 slides to assess intra-observer reliability (experimenter blind with respect to strain, sex, and age).
11. Estimates are individually corrected for histological shrinkage by determining the previously computed ratio between the brain volume at fixation and that after processing.
12. Estimates are individually adjusted for sex, age, epoch (Taylor strains vs. UTHSC Rww strains), and brain weight.