Bx52 microscope

Immunostaining method was described in a previous publication [52 (link)]. Detail information about the reagents in this part was shown in Table S1. Primary antibodies including anti-TH, anti-p-α-syn, anti-NF, and anti-GFAP were used either for midbrain or sciatic nerve. Then, secondary antibodies of Alexa Fluor 488 and 594 were incubated. Olympus FV1000 confocal laser scanning microscope was applied to acquire images.
For immunohistochemistry, brain slices were incubated with primary antibody of anti-TH. Number of TH⁺ neurons in SNpc of midbrain was assessed using optical fractionator (Stereo Investigator software, Microbrightfield Bioscience, Williston, VT, USA). All stereological analyses were performed under × 200 magnification of Olympus BX52 microscope (Olympus America Inc., Melville, NY, USA).

The following standard pre-processing steps were applied to all sections. The Olympus BX52 microscope with an Olympus DP72 camera back was used to image the optic nerve cross-sections using manual adjustments with 40x and 100x objectives, at 4140 × 3096 pixels. This resulted in a measured 40x objective resolution of 18.65 pixels/μm, and 100x objective resolution of 45.45 pixels/μm, we use 40x and 100x to indicate the images thus obtained respectively (Table 1). At 40x, imaging yields four to six raw images with varying amounts of overlap to cover the whole nerve. 40x was selected as the empirically optimal level of magnification for imaging. At 100x, imaging yielded 10 images per nerve, with areas randomly sampled from the whole nerve, so these images contain no overlap. The total area of the optic nerve cross-sections were determined by manually outlining the optic nerve border in ImageJ. The measured nerve area was used to determine the size of the counting frame for manual counting. A breakdown of the different datasets prepared and analyzed in this study is provided in Table 1, giving mouse model, number of mice, magnification, and number of images for each magnification.

Immunofluorescence and immunohistochemistry were performed following the protocols described in our prior study [22 (link)]. Cells or brain slices (frozen sections, 30 μm) were fixed in 4 % paraformaldehyde, blocked with 5 % BSA in PBST (0.3 % Triton X-100), and then incubated with desired primary antibodies at 4 °C overnight. The corresponding secondary antibodies were subsequently incubated for 1 h at room temperature. Image J (1.53q) was utilized for colocalization analysis as previously described [25 (link)], images for each channel were separately thresholded, and colocalization was defined as at least one pixel of overlap between the two channels. Neurite length was measured using Image J (1.53q). Stereology was performed for cell counting using the optical fractionator (Stereo Investigator 7, MBF Bioscience, Williston, VT). Every sixth coronal frozen section was collected for quantification. The regions of SNpc in the midbrain sections were outlined at low magnification (40×), and the counting frame size was 50 μm × 50 μm, with a sampling grid size of 100 μm × 100 μm. All stereological analyses were conducted under the 200× magnification of an Olympus BX52 microscope (Olympus America Inc., Melville, NY). The primary and secondary antibodies used in this study were listed in the Supplementary Table 1.