[Histonet] Frozen sections and cold acetone...

Fri Oct 26 10:09:02 CDT 2018

Tony, thanks for this. Our continued use of IF rather than IPOX is due to seemingly better signal/noise with IF. And now we have developed "rescue" techniques for IF on FFPE sections for cases in which the frozen portion is inadequate. Since we started that we have had more requests for IF of immunoglobulins on other FFPE tissues (liver, nerve, others) due to better signal/noise with IF vs IPOX of the same antibodies. 

On the question of cold vs room temperature acetone, I remember WAY back when we would cut frozens and immediately put them in -20C acetone for fixation. We got away from that and now put them in 2C acetone after they have dried for 10-30 minutes. Results are the same (at least for Ig and complement antigen staining). So, I'm going to try room temp acetone to see the results. Also some have mentioned they don't fix in acetone at all with good results. 

Tim Morken
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center

-----Original Message-----
From: Tony Henwood (SCHN) [mailto:tony.henwood at health.nsw.gov.au] 
Sent: Thursday, October 25, 2018 4:52 PM
To: Bryan Llewellyn; Morken, Timothy
Cc: histonet at lists.utsouthwestern.edu
Subject: RE: [Histonet] Frozen sections and cold acetone...

Hi Tim (and Bryan),

This is from a book chapter I wrote (though not published as yet), that might be useful:

"It is important that fixation has minimal effect on antigenic determinates, allowing recognition by appropriate antibodies. Fixatives can be physical or chemical in action. Physical fixatives, for example heating sections or cells on to slides, alcohol or acetone treatment, preserve tissues by dehydrating cells. Alcohol and acetone possibly also dissolve lipids from cell membranes facilitating antibody access."

 And

"With today's modern immunohistochemical methods being done quite successfully on formalin-fixed, paraffin-embedded sections, it is mystifying as to why immunofluorescence is still preferred for immunopathological renal and skin diagnosis. Immunofluorescence remains the most popular method of detecting immunoglobulins and complement deposits, with 83% of British laboratories using this technique (36). 
Technically, frozen sections are cut, dried, rinsed in buffer, optimally diluted FITC labelled antibodies are added to sections, incubated for 30-60 minutes, non-bound antibody is rinsed from the sections with buffer, the sections are coverslipped with a non-fluorescing aqueous mountant and finally viewed using a microscope equipped with an ultra-violet light source; overall quite a simple technique."

"The detection of tissue-bound autoantibodies is rendered difficult by the presence of abundant plasma proteins, which must be removed to avoid non-specific staining. This has resulted in a continuing popularity of frozen sections and immunofluorescence techniques in immunopathology, where they have been superseded in most other branches of immunohistochemistry by methods that are applicable to paraffin wax sections. In paraffin wax sections, the plasma proteins have been fixed in the tissues and must be removed by a digestion process rather than simple washing (37). Unfortunately, too much digestion may destroy tissue integrity and possibly remove the immune complexes, resulting in a false negative result. Furthermore, digestion conditions are difficult to standardize. The times required vary with the degree of fixation and section thickness (38). Frozen sections of non-fixed tissue, when used for the detection of bound-immune complexes, give clean localization with minimal background without the requirement for enzymatic pre-digestion. The plasma immunoglobulins, being loosely bound, if bound at all, to the surrounding tissue elements are easily dissolved in the warm buffer pre-incubation step. Bound immune-complexes seem to be unaffected."

36.	Furness PN & Boyd S (1996). Electron microscopy and immunocytochemistry in the assessment of renal biopsy specimens: actual and optimal practice. Journal of clinical pathology, 49(3):233-237
37.	Furness PN (2000). ACP Best practice No 160. Renal biopsy specimens. Journal of clinical pathology, 53(6):433-438.
38.	Lehman JS & Camilleri MJ (2013). Potential diagnostic pitfall in direct immunofluorescence evaluation for vasculitis in skin biopsies: fluorescent secretory granules within eccrine glands. Journal of cutaneous pathology 40:603-605

Regards 

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal 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: Bryan Llewellyn via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Friday, 26 October 2018 5:54 AM
To: Histonet
Subject: Re: [Histonet] Frozen sections and cold acetone...

Cold acetone, and cold ethanol, were used to fix tissues because they left enzymes unaffected and still demonstrable. This was in the early days of enzyme histochemistry. Pearse' Histochemistry: Theoretical and applied,3rd edition, volume 1, page 85 discusses it. I could send a scan if you wanted.

Bryan Llewellyn

Morken, Timothy via Histonet wrote:
> Can anyone give me a rational for using cold (refrig or freezer-temp) acetone to fix frozen sections? Or a rational for using RT acetone.
>
> This is for kidney or muscle bx frozens for immmunofluroescence or immunoperoxidase staining.
>
> Normally they air dry for at least 15 minutes (just waiting for frozen sectioning to be completed) before going into acetone. Just wondering if we can reduce complexity...
>
> I haven't seen anything saying why cold acetone is used, just instructions to do so. I always wonder about such things...
>
> Tim Morken
> Supervisor, Electron Microscopy/Neuromuscular Special Studies 
> Department of Pathology UC San Francisco Medical Center
>
> _______________________________________________
> Histonet mailing list
> Histonet at lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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