[Histonet] [Help] oil red O stain in fattty change liver

[Margaret Blount]

We cryoprotect our livers in 20% sucrose overnight at 4C after fixation in 4% paraformaldehyde, then freeze in thawing isopentane (freeze it over liquid nitrogen, lift clear and use it as it melts, but before it has melted completely). Store if necessary at -80C. Section onto charged (Plus) slides and stain in the normal way. I cannot see why yours are coming off, but suggest you try plus slides, then they won't. 

I can't think that the cryoprotection makes any difference to whether they fall off or not, but I think the sections are easier to cut after cryoprotection than without it. I have been given livers treated both ways.

Plus slides: Superfrost plus, available from all good histology suppliers; ours come from VWR.

Good luck.

Miss Margaret Blount
Histology Manager
Metabolic Research Laboratories
Level 4 Institute of Metabolic Science
Box 289, Addenbrooke's Hospital
Hills Road, Cambridge, CB2 0QQ

Tel 01223 769061/336079



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of kyoung LEE
Sent: 14 April 2011 15:07
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] [Help] oil red O stain in fattty change liver

Dear sirs.

I don't know whether I send this question to you.

For several weeks, I'm setting oil red O stain in fattty change liver (mouse).

Could you review my protocol?

1. Murine tissue,livers, is fixed with 10% NBF(10% neutral-buffered formalin).
2. Make frozen block 
3. Blocks were cut 5um in crystat and dried for 1~2H at RT. 
4. store at -20 
5. let it dry for 30mins at RT
6. immerse it in cold 10% NBF for 10mins
7. staining

After staining, my section were fallen off and seperated.

I search many protocols. Some suggest additional fixation is necessary as soon 
as cutting.
Others suggest prefixed tissue is not necessary additional fixation.

Plz send me your comments.
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet