Buffered glycerol is a versatile clearing and mounting medium for embryos. It is particularly useful for fluorescence-labeled specimens since "anti-fading" reagent can be supplemented with glycerol to increase the half-life of the fluorescence signal.

In general, buffered saline (e.g. phosphate, Tris or HEPES buffered saline) is added to glycerol to yield a 70%-80% glycerol solution). For fluorescent protein, we prefer PBS. For fluorescein (optimal pH is ~10), we use TBS pH 9.5.

4% n-propyl gallate (Sigma 02370) is added as an anti-fading reagent. To do this, dissolve 4 grams of n-propyl gallate in 80 mL glycerol by stirring in the dark overnight. In the following day, buffered saline is added to make the final volume 100 mL.

One drawback of clearing specimen with buffered glycerol is that the specimen would turn purple after extended light exposure. This photoreaction can be retarded by treating the specimen with acid solution (e.g. acid glycine or PBS adjusted to pH 2.0) before being transferred to glycerol. Furthermore, the BM Purple/NBT-BCIP stain would look relatively "dull" in glycerol (c.f. the brilliant blue color in epoxy or methacrylate monomer). However, one can use a mixture of ethanol and glycerol for clearing and mounting of BM Purple/NBT-BCIP stained specimens. We use a 1:4 Ethanol:Glycerol mix and allow the ethanol to evaporate further before putting on the coverslip.