#Results
Incubation time was a very strong factor in staining density and also affected the reliability of the homogenized tissue standards. On average across the brain areas measured, staining density doubled with each 30 min increment of incubation time (p < 2x10-16). There was also a incubation time by tissue thickness interaction effect, driven by the fact that differences between the two thicknesses (16 and 30uM) did not appear in the 30 min incubation time but were only apparent with longer incubation times (p = 0.00197) In addition the 30 min incubation time showed a lack of variation compared with the longer incubation times. Both of these facts indicate that the staining was incomplete at 30 min \ref{fig:Figure_1}. The relative effect size of tissue thickness was much lower than that of incubation time, at least when averaging across brain areas.