Supplementary Figure 2
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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 pre-optimization (BOPSS_0, D-F) and
post-optimization analysis (BOPSS, G-I). The blue arrows indicated examples
of reduced non-specific detection in post-optimization analysis. The manually
counted puncta were marked in black and labelled with yellow numbers with
Cell Counter (Image J) (J-L). The orange and white arrows indicate examples
of overcounted and undercounted puncta detected by BOPSS compared with manual
counting, respectively. One-way ANOVA was performed to analyze the results of
single PLA. There is no significant difference among three quantification
methods (P value=0.113) (M). Two-way 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.
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