Images of in situ hybridization and immunostaining results were captured with a Spot 2 CCD digital camera using Spot Advanced image acquisition software (Diagnostic Instruments, Sterling Heights, MI) on an Olympus IX-70 microscope. Images were prepared as panels using Adobe Photoshop (San Jose, CA).
For comparing cell densities, all quantification was performed by an investigator blinded to the treatment condition. Cells expressing PLP mRNA were quantified using unbiased stereologic morphometric analysis (12 (link)) (Stereologer System from Systems Planning and Analysis, Inc., Alexandria, VA). Analysis was restricted to the corpus callosum region from the midline and extending laterally to below the cingulum in 15-μm-thick coronal sections. Using the Stereologer System, the specimen thickness contributes to the sampled volume so that measurements reflect cells/mm3. The unbiased stereologic method could not be used appropriately for conditions with relatively few cells of interest in any chosen category. Therefore, quantification of cells in the corpus callosum expressing PDGFαR or Ki-67 required counting all labeled cells and using the Spot 2 CCD camera and software to measure the area sampled, resulting in density units of cells/mm2 (12 (link)).
Quantification of corpus callosum myelination was estimated from MOG immunofluorescence detected with a Spot 2 CCD camera. Using Metamorph software, pixel intensity values were normalized between sections by thresholding to exclude values below the level of immunoreactivity in the dorsal fornix, which was selected as an adjacent white matter tract that is not demyelinated by cuprizone. The percent area of the corpus callosum (midline bilaterally to a point under the cingulum apex) with MOG immunoreactivity above the threshold level was then used as an estimate of the myelinated area.
Each category analyzed included three or more tissue sections per mouse and three or more mice per condition, except where larger sample sizes are noted in text and/or figure legends. One-way analysis of variance (ANOVA) with post hoc Tukey's multiple comparison test was used to determine significant differences among stages of disease progression or treatment. Unpaired Student t-test was used to compare between FGF2 genotypes in nonlesioned mice. Significance of an FGF2 genotype effect across multiple treatment conditions was calculated using a two-way ANOVA. No statistical comparisons were made between mice with different genetic backgrounds (i.e. C57Bl/6 mice and FGF2 mice).
For comparing cell densities, all quantification was performed by an investigator blinded to the treatment condition. Cells expressing PLP mRNA were quantified using unbiased stereologic morphometric analysis (12 (link)) (Stereologer System from Systems Planning and Analysis, Inc., Alexandria, VA). Analysis was restricted to the corpus callosum region from the midline and extending laterally to below the cingulum in 15-μm-thick coronal sections. Using the Stereologer System, the specimen thickness contributes to the sampled volume so that measurements reflect cells/mm3. The unbiased stereologic method could not be used appropriately for conditions with relatively few cells of interest in any chosen category. Therefore, quantification of cells in the corpus callosum expressing PDGFαR or Ki-67 required counting all labeled cells and using the Spot 2 CCD camera and software to measure the area sampled, resulting in density units of cells/mm2 (12 (link)).
Quantification of corpus callosum myelination was estimated from MOG immunofluorescence detected with a Spot 2 CCD camera. Using Metamorph software, pixel intensity values were normalized between sections by thresholding to exclude values below the level of immunoreactivity in the dorsal fornix, which was selected as an adjacent white matter tract that is not demyelinated by cuprizone. The percent area of the corpus callosum (midline bilaterally to a point under the cingulum apex) with MOG immunoreactivity above the threshold level was then used as an estimate of the myelinated area.
Each category analyzed included three or more tissue sections per mouse and three or more mice per condition, except where larger sample sizes are noted in text and/or figure legends. One-way analysis of variance (ANOVA) with post hoc Tukey's multiple comparison test was used to determine significant differences among stages of disease progression or treatment. Unpaired Student t-test was used to compare between FGF2 genotypes in nonlesioned mice. Significance of an FGF2 genotype effect across multiple treatment conditions was calculated using a two-way ANOVA. No statistical comparisons were made between mice with different genetic backgrounds (i.e. C57Bl/6 mice and FGF2 mice).
