I am trying to perform confocal 20X Z-stacks for the entire z-axis of 2-3mm thick, coronal adult mouse brain slices using 85% glycerol as mounting solution. I have been using a blutack to rest a coverslip on a slide with the slice. I have never had trouble with hydrogel embedding using a dessicator and mineral oil, or clearing using a hybridization oven and daily washes.
Normally, I can only see approximately 800um deep into the slice if I don't preincubate the section in glycerol (the objective lens can be lowered further, but it doesn't give a good signal). The other day, I left a slice in a 15mL tube containing glycerol for about 36 hours, and managed to get 2000um deep (I am using a fancy objective lens designed for SCALE meant to have a large working distance).
Does anyone know of factors or methods which increase or decrease the depth to which one can image in thick sections of cleared tissue?