Supplemental Methods and Materials
Supplemental Methods and Materials
Supplemental Table 1. Human Subject information
Supplemental Table 2. ROI, sampling areas and counting areas Supplemental Figure. 1. Sampling procedures for PLABF. Luxol fast blue/cresyl violet staining was performed to discriminate between white matter and grey matter (AC). An outline of ventral striatum subterritories (D) was drawn based on the stained results of each sample. Overlapping the sampling grid (E) and the outline (D) divided the brain section into several evenly distributed sampling areas (F and G). Within the NAcc, the ROI in this study (G), one counting locus (indicated by * bounded by purple frame in the inset) was selected in each sampling area (indicated by the blue frame in the inset) and the 40x image of this counting locus was exported for quantification (H). A section of (H) is shown at high magnification (I). Scale bar, AC, 5 mm; HI, 50 µm. Supplemental Figure. 2. Quantification of PLA signal with BOPSS and
manual counting. Three randomly selected areas in a full counting image of each
PLA condition, single (A), dual (B) and negative PLA (C) (from PI12277) were
quantified with BOPSS or manually (four times independently). The puncta counted by BOPSS were marked in
red in the representative images of preoptimization (BOPSS_0, DF) and
postoptimization analysis (BOPSS, GI). The blue arrows indicated examples of
reduced nonspecific detection in postoptimization analysis. The manually
counted puncta were marked in black and labelled with yellow numbers with Cell
Counter (Image J) (JL). The orange and white arrows indicate examples of
overcounted and undercounted puncta detected by BOPSS compared with manual counting,
respectively. Oneway ANOVA was performed to analyze the results of single PLA.
There is no significant difference among three quantification methods (P
value=0.113) (M). Twoway ANOVA was performed to compare the quantification
results for dual PLA and its negative control, which had the same PLA condition
as dual PLA but omit one primary antibody (N). The interaction accounts for
approximately 2.5 % of the total variance and is considered not significant (P
value=0.069). Both quantification methods (accounts 33.9 % of the total
variance, P value is <0.001) and PLA conditions (accounts 41.6 % of the
total variance, P value is <0.0001) have significant effect on the
variation. Bonferroni’s multiple comparison were performed to compare BOPSS and
other methods (M and N), **** multiplicity adjusted P value <0.0001, **
<0.01.
