[Histonet] Safranin O staining

[Victor Wong]

Hi Ray,
 
Thank you for your suggestion and the paper.  There is a wonderful staining with safranin O compared to our staining that was very faint, nearly as unstained with safranin O (there was staining with fast green and hx).
 
I dealed with sample as pellet in Falcon tubes, after experiment by my colleaque.  As far as I know, we isolated mesenchymal stem cells from marrow and cultivated in chondrogenic medium for days.  We don't have a pellet positive control but including a section with cartilage is useful.
 
I really concerned that high temperature during melting the 2% agarose may be deletrious to the staining.  As suggested, I will extend the fixation time.
 
Best Regards,
Victor
 
 

From: "koellingr <@t> comcast.net" <koellingr <@t> comcast.net>
To: Victor Wong <vhlwong <@t> yahoo.com> 
Cc: histonet <@t> lists.utsouthwestern.edu 
Sent: Monday, February 18, 2013 10:42 PM
Subject: Re: [Histonet] Safranin O staining


Victor, I completely agree with Tony and Renee's methodology assessment but I would also ask you to look at your induced chondrogenesis model (I've never done this but have done many, many other cell culture models).  If you go to this paper http://biotech.korea.ac.kr/bk21/bbs/upload/bk21bbs_cur_165_0.pdf in Tissue Engineering they describe in detail exactly what you are trying to do, if I read your question correctly.  Their pictures of safrinin 0 staining of pelleted cells from culture could be described as "light staining" if you compare them to the red/orange color what we all know safranin o looks like on articular surfaces.  I think that is a POOR positive control for their experiments but that's just me.  In-vitro cultured cells prepared for histology will seldom  stain the same as in-vivo material prepared for histology.  And they had to hit cells pretty hard, 5ng TGF to see some staining.  Have no idea what your cell culture
 methodologies are but what I think you are doing is all in that paper and you can see the results in the Safranin o pictures.  There are ways to get better, more similar, positive staining controls then they used.
 
Ray Koelling
Research Scientist
University of Washington
Seattle


From: "Victor Wong" <vhlwong <@t> yahoo.com>
To: histonet <@t> lists.utsouthwestern.edu
Sent: Sunday, February 17, 2013 6:34:12 PM
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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