Dear Dr. Jordan Donohue

Excess water/PBS in the agarose block can lead to cracking upon freezing and sectioning. The water expands during freezing and exerts pressure on the agarose, causing it to fracture. So, it becomes essential to remove as much water/PBS as possible from the tissue before embedding. This can be achieved by blotting the tissue with absorbent paper or by swirling it in the agarose solution to allow the agarose to absorb excess liquid.

Use 2-3% low melting point agarose in 1X PBS. Ensure that the agarose is fully dissolved and that there are no air bubbles in the solution before embedding. Using agarose that is either too concentrated can make the agarose brittle or too dilute may not provide sufficient support leading to cracking.

Regarding the vibratome, you mentioned that the vibratome is pretty old but seems to be working fine. Try adjusting the vibratome settings to optimize sectioning. Lower the speed and increase the oscillation frequency. Experiment with different settings to find the optimal parameters for your tissue and agarose. Too high a speed or too low an oscillation frequency can stress the agarose block and cause it to crack. Ensure the blade is sharp and free of nicks and should be changed after each use. Verify that the blade is properly aligned with the specimen tube.

Best,