Hv c20

PFA-fixed cryopreserved brains were sectioned at 30 µm thickness using a sliding microtome (Microm HM 450, ThermoFisher, Waltham, MA). For SNpc cell counting studies, immunohistochemistry for tyrosine hydroxylase (TH) was performed using a VECTASTAIN Elite ABC-HRP kit (PK-6101) with rabbit anti-TH antibody from Novus Biologicals (NB300-109, 1:1,500). TH⁺ neurons and TH⁻ neurons (Nissl⁺) were counted in parallel using an unbiased computer-assisted stereological platform (Stereo Investigator software, MicroBrightfield, Willison, VT) connected to an Axiophot photomicroscope (Carl Zeiss) with motorized stage (Ludl Electronics) and camera (Hitachi HV C20). For immunofluorescence analyses of TH/pS129-αSyn, stained sections were imaged at equal exposure settings and total TH⁺ neurons were counted for each image. pS129-αSyn⁺ and TH⁺ (double-positive) cells were then counted and divided by the total number of TH⁺ cells. For pTBK1 and γH2A.X analyses, images were acquired using a Zeiss LSM 880 confocal scanning microscope and quantification of colocalization or intensity was performed using the Zen software (Carl Zeiss).

The number of DAPI-labeled nuclei in each scleral whole mount quadrant was quantified using Stereo Investigator software (MicroBrightField, Williston, VT) integrated with an ECLIPSE E600 microscope (Nikon, Japan) with a 3-chip charge-coupled device (CCD) color video camera (HV-C20, Hitachi, Japan) and motorized stage and microcator attachment (Heidenhain EXE 610C, Schaumburg, IL). All DAPI-labeled cells with the elongated shape and ovoid nucleus typical of fibroblasts were counted. Approximately four regions, each 460 µm × 300 µm, were quantified in each quadrant, comprising approximately 4% of the scleral area in a specimen.
Within each designated region, observations were made at three depths through the entire scleral thickness, with each depth comprising a zone approximately 10 µm thick. One depth was near the initial scleral edge, one in the mid-sclera, and one at the opposite scleral edge. Observations made at each of these three depth designations were not significantly different, and therefore, the average is presented. The density of cells per unit area (in DAPI-labeled tissue) and the number and percent labeling positively for Ki67 were analyzed by scleral region (from close to the peripapillary area to the equator), by mouse strain, and by experimental glaucoma compared to control.