Hello. I am used to paraffin protocols, but now I need sections of mouse heart from frozen tissue due to not having paraffin equipment. My protocol is:

Remove the heart from the animal and perfuse with 5mL PBS;

Put the hearts into Falcon Tubes with 5mL 4% PFA at 4°C for 48 hours;

Blot them carefully in paper towel (kim wipe) and perform a sucrose gradient (10, 20 and 30%) until they sink;

Blot them again and freeze in OCT using Isopentane in liquid nitrogen;

Wrap each block individually in aluminium foil and leave at -80°C until cutting (up to one week);

During cutting, I always use a new blade, but not the anti-roll, as the machine is from another lab. Cutting temperature is usually -19 to -21°C and thickness are 8µm;

I look under the microscope and they seem ok, however after HE staining the morphology is ruined to the point that nuclei are barely stained.

I tested spleens under the same protocol (processed together with the hearts) and, although they could be better, they look much better than the hearts.

Currently I am trying to find any reasons why this could be happening.