[EXTERNAL] RE: [Histonet] Question regarding dehydration

[Elizabeth Chlipala]

Julio

4 to 5 hours a station is way to long for processing samples of this size, regardless of what type of tissue or species they are from.  Graduated alcohol dehydration is better.  I would process samples of that size (unless it was bone, fat or skin) for no longer than 45 - 60 minutes a solution for manual processing.  

Here is what a typical processing cycle would look like once the tissue has been adequately fixed.

50% alcohol  (you want to start here if you are working with small animal tissue such as mice and rats)
70% alcohol 
80% alcohol
95% alcohol
100% alcohol
100% alcohol
Xylene
Xylene
Paraffin
Paraffin
Paraffin

I hope this helps, you may be able to get sections by trimming into the block and then soaking them on wet ice for some time (possibly an hour or so).

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Julio Benavides
Sent: Friday, February 13, 2015 8:43 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [EXTERNAL] RE: [Histonet] Question regarding dehydration

Thanks Ryan. Tissu sections where thin, something about 2 and 3 mm. I think they were well fixed (buffered formalin) nut the problem was the dehydration. to go from formalin straight into 100% ethanol looks a bit too drastic to me.

Thanks for your thoughts

Julio

On 13/02/2015 16:44, Roy, Ryan wrote:
> Its well documented in the literature that using graded alcohols in processing is advantageous to prevent hardening of the tissue.
>
> How thick are the tissue section cut that are being processed? It is also well documented that sections should avoid being cut thicker than 3-4mm as this prevents the penetration the fixative as well as the other reagents.
>
> 4 hours in each reagent seems excessive... ask other people too since I have limited experience.
>
>
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu 
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Julio 
> Benavides
> Sent: Friday, February 13, 2015 10:19 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [EXTERNAL] RE: [Histonet] Question regarding dehydration
>
> Hi Ryan,
>
> thanks a lot for your thoughts. These blocks were processed elsewhere and sent to us for the cutting and staining. Tissues were dehydrated in five consecutive baths of ethanol 100%, 5 hours each (manual processing). Then, they went to xilene (three baths, 4 hours each) and paraffin (two batch, 1h30´ each). Truth is that I have never seen such processing before. In our lab, with automatic processing, we begin with 60% ethanol and go to 70, then 95 and then 100 before ethanol/xylene.
>
> I was wondering if anybody has used the 100% ethanol processing before and which was the influence over tissues.
>
> Thanks
>
> Julio
>
>
> -------- Forwarded Message --------
> Subject: 	RE: [EXTERNAL] [Histonet] Question regarding dehydration
> Date: 	Fri, 13 Feb 2015 09:06:47 -0500
> From: 	Roy, Ryan <Ryan.Roy <@t> va.gov>
> To: 	'Julio Benavides' <j.benavides <@t> eae.csic.es>
>
>
>
> If the tissue is fatty it will blow apart on the water bath. If the H and E staining is poor its probably the processing.
>
> What is the exact procedure for your process including time that the tissue was in each reagent? I would include this in a message to all of histonet as well as some may more knowledgeable than myself.
>
> Also, I would recommend using a 95% solution prior to the 100% alcohol (s) if not a 70-80% and 95%.
>
>
> Ryan Roy HTL (ASCP)
> Manchester Veterans Affairs Medical Center Manchester New Hampshire
>
> Disclosure: The content of this email does not represent the views or 
> opinons of the VA
>
>
>
>
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu 
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Julio 
> Benavides
> Sent: Friday, February 13, 2015 4:26 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [EXTERNAL] [Histonet] Question regarding dehydration
>
> Hi there,
>
> we are having problems trying to cut some embedded samples (they crumble in the bath and the few cuts we manage to get into HE are crap). These are formalin fixed samples (bovine foetal and placenta samples) which went straight from formlin into 100º ethanol for the dehydration before clearing. I was wondering if such drastic dehydration step (no 60º, 70º or 90º ethanol before the 100º) could have damage the tissue. Has anyone have a similar issue before? do you think the samples are ruined for histology?
>
> Thanks a lot for your help
>
> Regards
>
> Julio
> Instituto de Ganaderia de Montaña
> Spain
>
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