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1. Buffered Glycerol
To make 50ml: Mix 25 ml glycerol, 5 ml 10X PBS, pH 7.4, 20 ml
dH2O. Immerse cells or tissue in three changes to ensure
penetration. Mount with fresh buffered glycerol, coverslip and seal with
Refractive index = 1.42
2. Methyl Salicylate
Use without buffering. Immerse cells in 3 - 4 changes to eliminate
residual H2O. Adjust time to insure adequate water removal.
Mount with fresh methyl salicylate and seal coverslip with nail polish.
(This procedure has been worked out for clearing
thick slices of rat brain tissue (~0.5 mm thick) previously fixed with
1. Wash 2x in fresh PBS, 5 min each.
2. Wash 2x in dH2O, 5 min each.
3. Dehydrate with a graded ethanol series: (20%,
30%, 50%, 70%, 95%), 5 min each
4. Finish dehydration with two100% ethanol washes,
5 min each.
5. Begin clearing by soaking in methyl salicylate,
6. Change for fresh methyl salicylate, soak
6. Mount sample in fresh methyl salicylate,
coverslip, seal with nail polish.
Refractive index = 1.54
alcohol - benzyl benzoate)
benzyl alcohol (Sigma B-1042)/ benzyl benzoate (Sigma B-6630) 1:2 ratio
with graded MeOH series before sinking tissue into BABB as follows:
MeOH (25 % MeOH / 75 % PBT (PBS with 0.1% Tween-20) for 5 min
MeOH, 5 min,
MeOH, 5 min,
MeOH, 2x 5 min each
MeOH - 50% BABB, 2x 5 min each
“Sink” tissue into BABB solution ON at RT.
Mount in barest minimum droplet of BABB, coverslip, seal with nail
Refractive index = 1.51