[Histonet] Porcessing FFPE tissue without alcohol??

[Elizabeth Chlipala]

Carl

We have tried multiple procedures, there are procedures that use a combination of 2% osmium and 5% postassium dichromate, ones that call for shorter periods of time in Osmium and then periodic acid rinse post osmium - this is referenced in Freida's book - Histotechnology a Self-Instructional Text.  We ultimately settled on a modification of different methods.  We use a 1% osmium solution for 24 hours rinse well in water and process same day short cycle (20 minutes per station with propar instead of xylene).  I'll post some images and our procedure on the block.  Sectioning and staining of these samples is tricky and we have found at least in our hands that the tissue is very friable (liver primarily, sciatic nerve and muscle samples turned out better) and does not stay on the slides well so we have not been able to even counterstain with H&E without considerable tissue loss.   Sections can be a bit uneven which is OK for routine viewing via a microscope but not good for whole slide scanning and image analysis applications.  I have pics of all of the problems associated with this protocol along with some really nice ones.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz at premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-----Original Message-----
From: Hobbs, Carl [mailto:carl.hobbs at kcl.ac.uk] 
Sent: Saturday, March 26, 2016 12:56 PM
To: Elizabeth Chlipala; Joanna; Rene J Buesa
Cc: histonet
Subject: Re: [Histonet] Porcessing FFPE tissue without alcohol??

Thanks, Liz.
If you look at fat all the time, using Osmium.....you then are not sure if you use K-dichromate?
I am a tad confused

Also....why not trim the block too much?

Best wishes,

Carl
NB: Rene stated that I wouldn't be able to use any other fat stains...that's the point, Rene.
I don't need any other.
I commit to Osmium.

Yep...there are many variations for fat staining.
Imho...most are histochemical mythology.
Osmium "stains" fat...histologically.
As do  the conventional fat-soluble dyes when using frozen sections.
I would only listen to alternatives/disagreements from JKiernan.

I am still waiting for the next "generation" Histo person.....
Cook, Kiernan.....who are the other Seminals??

Enquiringly

Carl

________________________________________
From: Elizabeth Chlipala <liz at premierlab.com>
Sent: 26 March 2016 15:58
To: Joanna; Rene J Buesa
Cc: Hobbs, Carl; histonet
Subject: RE: [Histonet] Porcessing FFPE tissue without alcohol??

We use osmium post fixation to look at fat all of the time in mouse liver, nerve and muscle samples.  It works well, sample size needs to be thin, samples are friable and can crack easily.  We use a specific procedure for this it includes potassium dichromate I think, I'm at home but on Monday I can send the reference.  One more thing don't trim into the block too much.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
liz at premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504
________________________________________
From: Joanna via Histonet [histonet at lists.utsouthwestern.edu]
Sent: Saturday, March 26, 2016 9:20 AM
To: Rene J Buesa
Cc: Hobbs, Carl; histonet
Subject: Re: [Histonet] Porcessing FFPE tissue without alcohol??

How about Sudan Black stain?

> On Mar 26, 2016, at 4:32 AM, Rene J Buesa via Histonet <histonet at lists.utsouthwestern.edu> wrote:
>
> The only problem I see is that the fat will be preserved, as you 
> wrote, as a black osmium oxidate but you will not be able to use any 
> "standard" fat stain; otherwise it will work.René
>
>    On Friday, March 25, 2016 2:41 PM, "Hobbs, Carl via Histonet" <histonet at lists.utsouthwestern.edu> wrote:
>
>
>
>
> Fix the tissue in Formalin, wash well in dw, then place very small 
> pieces into Osmium tetroxide solution ( std soln for TEM post-fixation) Processing to Pwax as usual.
> Basically, you will see lipids as black ( oxidised osmium) That's the 
> only way to demonstrate solvent- soluble lipids, using Pwax processing.
> Sure, there are caveats but, in the main...it will be Ok, imho.
> I invite comments as I may be doing exactly this very soon, to count myelinated nerve fibres in a sciatic nerve.
>
>
>
>
>
> Carl Hobbs FIBMS
> Histology and Imaging Manager
> Wolfson CARD
> Guys Campus, London Bridge
> Kings College London
> London
> SE1 1UL
>
> 020 7848 6813
> _______________________________________________
> Histonet mailing list
> Histonet at lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
> _______________________________________________
> Histonet mailing list
> Histonet at lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet

_______________________________________________
Histonet mailing list
Histonet at lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet