Hello,
Does anyone have any advice on getting good frozen sections from tissue that has been stored in formaldehyde for prolonged periods?

I am currently cutting fish gonads on a cryostat. This could potentially save a lot of time and expense compared to wax histology. My samples have been stored in 4% buffered formaldehyde for several months. Due to the nature of my sampling, this is difficult to avoid. I have been cryoprotecing the tissue with a mixture of DMSO and sucrose, and then freezing in a bath of hexane cooled with dry ice. I am taking 6-10um sections which are then stained with H&E and viewed with a light microscope. For the female gonads, this is working quite well, the morphology is good and cryprotection seems to keep freeze damage to a minimum. However, for the testes the results are not so good. Sections are cracked and morphology is really poor. When the same tissue is cut using wax histology the sections are fine. Rinsing the tissue well in PBS to remove excess formaldehyde before sectionning on the cryostat helps, as does cryprotecting for longer periods (overnight) but cracks are still visible. Could prolonged storage in formaldehyde render the tissue less permeable to the cryoprotectant perhaps? Any suggestions/remedies would be much appreciated as I am running out of things to try!

Best wishes,

Deirdre

 

_______________________

Deirdre Brophy

Dept of Life Sciences

GMIT

Dublin rd

Galway

Ireland