BBBA (aka BABB or Murray's clear) is made by combining 2 parts benzyl benzoate and 1 part benzyl alcohol. Alternatively, a mixture of 3 parts benzyl benzoate and 2 parts benzyl alcohol is used for clearing leech embryos. This clearing medium is an effective clearing medium for the yolky early-stage leech embryo. The specimen, however, shrinks significantly in BBBA. Its utility is also limited since many staining/labeling reagents does not survive well in this medium. We are comfortable about using BBBA with BM Purple (a chromogenic alkaline phosphatase substrate), DAB (a chromogenic peroxidase substrate), and fluorescent dye-conjugated secondary antibody.

To clear the specimen, the specimen is dehydrated by passing through a alcohol series (methanol, ethanol, or propanol are all applicable, however, methanol or ethanol is preferred), e.g. 1X 50% EtOH, 1X 70% EtOH, 1X 80% EtOH, 1X 90% EtOH, 1X 95% EtOH, 2X 100% EtOH. An extended incubation (>30 minutes) in absolute alcohol is recommended. Replace alcohol with BBBA. Once alcohol and BBBA have reached the equilibrium state, the specimen is ready for mounting into a slide for observation. Long-term storage of specimen in BBBA is not recommended. After imaging, it is advisable to return the specimen back to absolute alcohol for storage.

Note that BBBA can dissolve most plastic material over time. Store BBBA is a glass bottle or a PP container. Also, use silicone-well slide or glass welled slide for microscopic observation.