[Histonet] Help with fixed-frozen tissue sections

[Alfred Penzo Mendez]

Dear Histonet members,

for a few years I have done IHC on fixed-frozen mouse embryos with good success. A few days ago I moved onto adult tissues (liver and pancreas) and have met diffulties getting sections of good qualitiy. Here is how I proceeded:

- Pancreas and liver were fixed whole, in Zinc-formalin (polysciences) for 2 hours at room temp.

- Fixative was washed in PBS, 4 X 10 min.

- cryoprotection: 15% sucrose PBS until in sinks (~1 hour), followed by 30% sucrose o/n then 1 hour in 1:1 sucrose/OCT. 

- Embedding: I blot excess sucrose/OCT with paper and then embedd in OCT. I like to place my mold on a thin plastic raft (the type that you use to wheigh powders)which I float on liquid nitrogen. This way the bloc freezes in 1 to 2 minutes.

- Sectioning: I cut at 7 um. I used to cut embryos at -20 oC, but found that liver/pancvreas won't cut above -15 oC (they become hard and I get Venitian blind artifact).

- H&E stain: I aird dry my sections 1 hour, then fix 10 minutes in Zinc-formalin, wash in PBS and stain regressively with Gill's II. I differentiate in 0.12 N (1%) HCl in 70% ethanol, blueing is done in 0.25% ammonia. 

- THE PROBLEM: is twol fold. first the architectur of the tissue is damaged, liver cells become "separate" so structure of parenchyma and lobules is lost. Something similar happens to the acinar pancreas, with loss of shape of acini. I suspect this is tissue compression from ice, which I don't understand siince I cryoprotect (I think) properly. Second is that my section tend to detach during the H&E, specially during diufferentiation step they detach. I never had this problem whith embryos, which were barely fixed (20 min. or less). 

I put a picture of liver under "alfredo-liver-artefact"; I'd greatly appreciate any input.

Many thanks (and apologies for the lengthy post),

Alfredo Penzo
Abramson Family Cancer Research Institute
Philadelphia, PA.