Dear All,

I am doing IF over mouse spleen recently. But when I sectioned the tissue into slices, I met some problems.

I fixed the mouse whole spleen with 4% PFA in PBS at 4C for 5, 8, and 18 hours respectively. Also tried to fix in 10% formalin (Home made) for 8 hours at room temperature. Then I dehydrate the spleen with 30% sucrose in PBS overnight, or for 24hours, and I did observe all spleens sunk to the bottom of sucrose solution. Finally, I put the spleen to OCT and immediately froze at -80C fridge. The mold I used here was folded by tin foil and I froze the sample by sitting the mold to the metal plate of the -80C fridge.

But first, when I sectioned the tissue and touched the slice with cyto glass, the OCT adhere to the glass smoothly, while the only the rim of the tissue adhered to the glass and the center part formed a vault. The bubble like center part cannot settle down to the glass surface until I take the slide and warmed the back of the slide with my finger.

Secondly, many tiny holes formed when I leave tissue sections at room temperature after a few seconds.

I will appreciate any suggestion from you!

Bests

Binbin