Re: [Histonet] Porcessing FFPE tissue without alcohol??

[Tony Henwood (SCHN) via Histonet]

You might find these articles to be useful

Tracy, R. E., & Walia, P. (2002). A method to fix lipids for staining fat 
embolism in paraffin sections. Histopathology, 41(1), 75-79.

Turello, R., Snyder, D., & Hartman, H. A. (1984). A modification the osmium 
tetroxide post-fixation technique for the demonstration of extracellular lipid 
in paraffin-embedded tissue sections. Journal of Histotechnology, 7(2), 75-77.


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Hobbs, Carl via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Saturday, 26 March 2016 5:30 AM
To: histonet
Subject: Re: [Histonet] Porcessing FFPE tissue without alcohol??



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
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Re: [Histonet] Porcessing FFPE tissue without alcohol??

[Elizabeth Chlipala via Histonet]

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 
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
l...@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.ho...@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

Alsowhy 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 
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
l...@premierlab.com

Ship to address:

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

From: Joanna via Histonet [histonet@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 
>  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" 
>  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
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> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
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> 

Re: [Histonet] Processing FFPE tissue without alcohol

[Dessasau III, Evan via Histonet]

Will they be able to do IHC stains?

-Original Message-
From: Wanda Shotsberger Gray via Histonet 
[mailto:histonet@lists.utsouthwestern.edu]
Sent: Saturday, March 26, 2016 12:56 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Processing FFPE tissue without alcohol


While the tissue will still go through alcohol, have you considered preserving 
the fat with osmium tetroxide prior to routine processing? This turns the fat 
black, but it is retained in the tissue.


Hi Histonet, is there a way to process tissue for paraffin embedding without 
using alcohol? One of the labs that send their processing to us will be doing a 
study examining fat. I told them if they want to look at the fat they will have 
to cut frozen sections but I'm being ask again about processing without the 
alcohol. So I said I would ask around.

Please let me know what you think.

Thank you for your help!

E-van



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[Histonet] Cytology fixative

[McCabe, Sara via Histonet]

Wondering what everyone is using for their "wet" respiratory cytology 
specimens(bronch specimens, BAL, brushings etc)
Currently we have our microbiology department use mucolytic agent.  We also use 
cyto rich red for our brushing specimens. We are having a "drying out" effect 
and are wondering if these are causing this?
Any info would be greatly appreciated!
Sara J. McCabe, HT(ASCP)CM
Senior Histotechnologist
Penn Highlands DuBois
100 Hospital Avenue
DuBois, PA 15801
814-375-3264 Telephone
814-375-3784 Fax
sjmcc...@phhealthcare.org
www.phhealthcare.org

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