[Histonet] Safranin O staining

[Victor Wong]

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] On Behalf Of Victor Wong
Sent: Monday, 18 February 2013 1:34 PM
To: 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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