Agarose Cube for Cleared Samples

Procedure

  1. If the samples are already dehydrated, must do a rehydration step:

    • 100% Methanol - 10 minutes.

    • 75% Methanol - 5 minutes.

    • 50% Methanol - 5 minutes.

    • 25% Methanol - 5 minutes.

    • PBS - 3x 5 Minutes.

  2. Embed the sample in an agarose cube.

  3. Dehydrate with methanol gradient.

    • 25% Methanol - 5 minutes.

    • 50% Methanol - 5 minutes.

    • 75% Methanol - 5 minutes.

    • 100% Methanol - 10 minutes.

    • 50/50 BABB/Methanol Overnight, mutating at 4 degrees.

  4. BABB.

Notes and Cautions

  • If you go directly from 100% methanol to BABB, the agarose cube will collapse.

  • Numbers below are for smaller samples.

  • Duration may need optimization on larger specimens.

  • Percentage?

  • Low-melting temperature?