Standardized Immunofluorescence and Stereology Protocol

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.