[Histonet] Safranin O staining

[Tony Henwood (SCHN)]

Hi Victor,

I suppose the take home message from Russ's paper (God rest his soul - we do miss him) is that heat will affect some stains so extrapolating to routine processing temperatures, one could suppose that the deleterious effect would be more pronounced. To protect the cells, I would extend the fixation time and look at fixing before preparing the agar pellet. Or use the thrombin method instead (ask your Haematology department for expired thromboplastin from their clotting tests, use expired plasma from blood bank, add a few drops of 1% calcium chloride and a clot should form in 20seconds or so. Add NBF, fix for longer than an hour (4hr to overnight is better. And process as usual. Since the clots tend to be only about 3mm in greatest dimension (depending on the volume of thromboplastin and plasma used, a short gentle program will suffice (5 changes of alcohol, 20min each: 2 changes of xylene 30min each, 3 changes of wax, 30 min each).

Alternatively you could mix the pellet with some egg albumin, add an equal volume of 10% formalin in alcohol, a nice clot will form, fix for a few hours and process from alcohol (Based on the paper, with modifications, by Yamamoto et al (1985) Am J Clin Pathol 83:409-414).

Interstitial glycosaminoglycans and other mucins are also water soluble so fixing in formal alcohol should retain more of these "mucins" than aqueous NBF fixation (Nathan, & van Deth (1983) Pathology 15:301-4)

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

From: Victor Wong [mailto:vhlwong <@t> yahoo.com]
Sent: Monday, 18 February 2013 6:36 PM
To: Tony Henwood (SCHN); histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Safranin O staining

Dear Tony,

Thank you for your prompt reply and the paper.

I do fix the cell before processing in agarose and after embedding in agarose.

It is inevitable to process in wax at higher than 45C as it is set by the routine workers.

It is the first time I worked on cell block precessing.  Do you have protocol of processing chondrogenic pellet by tissue processor?  Any adjustment to my procesing and staining protocol?

I tried to stain the pH1.0 alcian blue for 45 minutes and overnght but unremarkable staining (faint staining) was observed.  Do you think glycosaminoglycans secreted by chondrogenic pellet can be destroyed by heat?

Best Regards,
Victor



From: Tony Henwood (SCHN) <tony.henwood <@t> health.nsw.gov.au>
To: 'Victor Wong' <vhlwong <@t> yahoo.com>; "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, February 18, 2013 11:45 AM
Subject: RE: [Histonet] Safranin O staining

Victor,

Heat can affect the way tissues and cells bind to dyes. I have attached an article that hints at some of these problems (Allison, R. T. and Bryant, D. (1998)'Effects of Processing at 45 C on Staining', Biotechnic and Histochemistry 73:3,128 -136).

I have also found that some antigens are adversely affected by heat. CEA immunohistochemistry was decreased in cytology cell blocks made with molten agar compared to cell blocks made with fibrin clot. In both cases cell blocks were fixed in NBF after being made. Formalin seemed to protect the antigens in fibrin clots from subsequent heat damage during processing, whereas with agar cell blocks, the heat damage was already done.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu<mailto:histonet-bounces <@t> lists.utsouthwestern.edu> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu<mailto:histonet-bounces <@t> lists.utsouthwestern.edu>] On Behalf Of Victor Wong
Sent: Monday, 18 February 2013 1:34 PM
To: histonet <@t> lists.utsouthwestern.edu<mailto:histonet <@t> lists.utsouthwestern.edu>
Subject: [Histonet] Safranin O staining

Hi all,

I am working on induced chondrogenesis in cell culture.  After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block.  When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure.  I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining.  Here is my protocol:

1. Weigert Iron Hx for 2 min
2. Acid alcohol,Scott Tap and wash
3. 0.02% fast green for 2 min
4. 0.1% acetic acid for 1 min
5. Sigma's Safranin O for 5-10 min
6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount

I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable.  I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose.  Are there other factors affect the staining. Any comments are welcomed.

Thanks you in advance.

Victor
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