Axiskop2 fluorescence microscope

Single-channel/monochromatic images taken with a Spot cooled camera (Diagnostic Instruments) mounted on an Axiskop2 fluorescence microscope (Carl Zeiss) are directly imported in ImageJ64 (NIH) and converted into an 8-bit gray format. Adjust the threshold to the image as a whole (or regions of interest) to pick up signals to be measured. For comparative analysis, the same threshold values are applied to all images taken from all experimental groups. Apply the threshold adjustment to convert the image into a binary image. For fluorescence intensity analysis, measurement is automatically performed on the binary image for mean and integrated density. For cell counts, the Analyze Particles function is selected to automatically count the number of particles (soma or nuclei) and analyze particle properties (size and shape) and distribution. The measurement data were exported directly for statistical analysis.

A list of antibodies used for immunohistochemistry (IHC) is given below in “Antibodies used for ICC, IHC, and WM immunostaining.” Brain tissue from euthanized mice was embedded in optimal-cutting temperature compound (OTC) and frozen on dry ice. Fresh frozen tissue was sectioned into 14-µm sections using a Cryostat Leica CM3050 S instrument. Sectioned tissue was fixed for 10 min in 4% PFA and washed three times with PBS. For MBP and NG2 staining, tissue was permeabilized with 0.1% Triton X-100 for 2 min and washed three times with PBS. Triton X-100 was omitted for O1 staining. Tissue was blocked in 10% FBS in PBS for 30 min at room temperature. Primary antibodies were diluted in the same buffer for 1 h at room temperature followed by three washes in PBS. Tissue was probed with the appropriate FITC-conjugated secondary antibodies diluted in the same buffer containing 50 nM of proETX-594 for 2 h at room temperature, washed three times with PBS, and mounted in Vectashield mounting medium with DAPI (Vector Laboratories). For detection of MAL, a rabbit antiserum against the rat MAL amino acids 114 to 126 was used as previously described (60 (link)). Immunostained sections were visualized using an Axiskop2 fluorescence microscope (Carl Zeiss), and images were digitally acquired with a Spot cooled CCD camera (Diagnostic Instruments) mounted on the microscope.