I’m currently having issues with cutting thick sections (~1mm) or mouse / human pathological samples on our Leica vibratome. I’ve described my procedure below. As opposed to getting clean cuts, the blade seems to ‘drag‘ the tissue out of the agarose block. Unsure if this is a sign of a blunt blade, improper embedding, incorrect cutting frequency / amplitude or something else:

1) I first embed 4% PFA-fixed and PBS-washed sample in melted agarose (2.5-4%) in H20.

2) I then put it in the fridge to cool / set

3) I load the buffer tray with PBS (at RT) before trimming the agarose block to size and supergluing the agarose block to the platform

4) I cut with the following settings (frequency 7, amplitude 7), although I’m not sure what these mean!

If anyone has had a similar problem and knows how to get around this I would be extremely grateful. And any reliable resources for optimising vibratome cutting would be much appreciated. Thank you!