[Histonet] Paraffin embedded mouse embryos - too dry

Tue Apr 7 11:54:11 CDT 2009

Good morning.
A colleague of mine recently placed the 10% formalin fixed mouse embryos in 2% agarose, let them solidify, then process at 30 minutes per station under pressure/vacuum.  The protocol was published, but right now I don't have that information.  Hope this helps in some way!
Jen Anderson

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Andrea Grantham
Sent: Tuesday, April 07, 2009 7:40 AM
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Paraffin embedded mouse embryos - too dry

I sometimes get embryos in the lab for processing and here are the  
protocols that I use for embryos in these stages:

E12.5 -
70% Ethanol - 12 min
95% Ethanol - 12 min
100% Ethanol x 2 - 12 min each
Xylene x 2 - 12 min each
Paraffin - 30 min
Paraffin x 2 - 1 hr each

no vacuum or pressure used except for the last paraffin

E15.5 -
70% Ethanol - 2 hrs (or two changes for an hour each)
95% Ethanol - 1 hr
95% Ethanol - 2 hrs
100% Ethanol x 3 - 1 hr. eash
Xylene x 2 - 2 hrs each
Paraffin x 4 - total of 10 hrs

I use some vacuum and pressure - in the last Xylene and in the paraffins

These protocols were provided to my by Gayle Callis and I have  
modified them a little. They work great for me and I don't have any  
problems with the sectioning.

Good luck!

Andi

Andrea Grantham, HT (ASCP)
Senior Research Specialist
University of Arizona
Cell Biology and Anatomy
Histology Service Laboratory
P.O.Box 245044
Tucson, AZ 85724

algranth <@t> email.arizona.edu
Tel: 520.626.4415     Fax: 520.626.2097

"happy slicing and dicing and may all your stains work perfectly" -  
Paula Sicurello

On Apr 7, 2009, at 7:05 AM, Barbara Schormair wrote:

> Dear all,
>
> I'm having trouble with my paraffin embedded mouse embryos. We use  
> E12.5 to E15.5 embryos and we embed and cut the whole embryo. When  
> we try to cut, the paraffin sheets are fine, but as soon as we get  
> to the embryo, the tissue disrupts and tears. I also hear rasping or  
> scraping sounds when cutting throught the embryo, so the problem  
> seems to be that the tissue is too dry or too hard.
> Here is my current protocol:
> Dissect embryos from the uterus and amnion in 1x PBS, then fix  
> overnight at 4°C in 4% PFA. Transfer to 70% Ethanol and store at 4°C.
> After 2 days to several weeks, we continue with the processing as  
> follows:
> E12.5: 96% Ethanol for 15min, 100% Ethanol for 15min, Xylol for  
> 15min (I also tried 10min Xylol and 8min Xylol, but with the same  
> results).
> E15.5: 96% Ethanol for 30min, 100% Ethanol for 30min, Xylol for  
> 30min (I also tried 20min Xylol and 15min Xylol, but with the same  
> results).
> Then bring into Paraffin and leave overnight at 65°C, next day  
> embedding and cooling down on-5°C cooling plate. Store at 4°C.
>
> What is the problem? Storage in 70% Ethanol for too long? Xylol  
> incubation times too long or too short?
> What is the critical step - the ethanol incubation or the xylol  
> incubation - should I try different times in Xylol or also try and  
> change the Ethanol incubation times?
>
> Thanks in advance for any help!
>
> Best regards,
>
> Barbara
>
>
>
> -- 
> Dipl. Biol. Barbara Schormair
> PhD student Institute of Human Genetics
> Tel.: 	0049-89-3187-3953
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