The number of huTau donor cells, huTau recipient cells, Alz-50+ neurons (misfolding), and DAPI+ nuclei (to assess cell loss) in the EC, subiculum, and hippocampal formation of AAV-injected hemispheres was determined by counting all [GFP+], [huTau+/GFP−], and [Alz-50+] cells in the area of interest, which was outlined as region of interest (ROI) using the CAST software (Olympus). The cell numbers were counted in three to four brain sections per mouse and three to five mice per group. For DAPI+ nuclei counting in the EC, we outlined layers 1, 2/3, and 4/5 of the EC and performed stereological counting of 20% of the nuclei in the ROI, followed by extrapolation to 100%. Counting was done on an Olympus BX51 light microscope equipped with a 20× objective.
Cast software
Manufactured by Olympus
CAST software is a tool developed by Olympus for image analysis and processing. It provides users with a range of functionalities to capture, visualize, and analyze digital images.
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Lab products found in correlation
2 protocols using cast software
1
Quantifying Neurodegeneration in Tauopathy
2
Stereological Estimation of Thalamic Neurons
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The total number of neurons in the thalamus was estimated using an unbiased stereological counting method as previously described by West and colleagues (68 (link)) by a researcher blind to mouse genotype. Briefly, using an Olympus BH2 microscope controlled by CAST software (Olympus) the region of interest in each section was marked at low magnification (2× objective). Subsequently, neurons were counted using the 100× objective by stepping through the region of interest with predetermined steps in the x and y directions (300 µm in both directions) in a raster pattern. Due to shrinkage of the tissue after drying and dehydrating, the average tissue thickness was 12.67 µm, and we therefore chose a dissector height of 8 µm, a counting frame size of 240 µm2 and a section sampling fraction of 3 to obtain a sampling volume, at which we counted between 100 and 200 neurons in each animal. From these data, we calculated the total number of neurons in the thalamus of each animal. Neurons were distinguished from other cell types as described by Hou et al. (69 (link)).
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