[Histonet] (no subject)

[Yves Heremans]

Hi all,

I am looking for a good paraffin embedding protocol for rat liver and
subsequent albumin staining. Tissue specimens are about 0.5 cm3. The tissue
that I have embedded until now seems too dry: upon sectioning, the tissue
disintegrates and the paraffin comes apart from the tissue itself. Soaking
in ice water improves the sectioning but I want to avoid having to do this
in the future. I use Paramat and the following embedding protocol :

fix in 4% PFA for 18-20h at 4°C
1h in 70% ethanol
1h in 80% ethanol
1h in 96% ethanol
2h in 100% ethanol
overnight in 100% ethanol
2h in a 1:1 mix of 100% ethanol and histoclear
3h in histoclear
overnight in histoclear
2h in a 1:1 mix of histoclear and paramat
3h in paramat
3h in paramat

I have to include the overnight incubations since everything is done
manually.

I have tried shorter dehydration times (30 minutes in 85-95-100 and 100%
ethanol, 2 x 30 minutes in histoclear and 3 x 1h in paramat) but without
any improvement.

Yves
  

Yves Heremans, Ph.D.
University of Minnesota
Stem Cell Institute
Tel 612-625-0964  
Fax 612-624-2436  

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