[Histonet] Safranin O staining

[Victor Wong]

Hi Rene,
 
Thanks for your great suggestion.
 
Yes, I'll extend the fixation time.  Do you think the embedding with melting agarose will be deletrious to the safranin staining?  I just use a heating water bath to melt the 2% agarose without temperature control.  I checked for complete melting and it was not boiled actually. Can I go through routine automatic tissue processing instead of manually?
 
For Weigert staining, we had a strong nuclear staining compared with faint or unstained red colour.
 
Any enlightenment is welcomed.
 
Best Regards,
Victor
 

From: Rene J Buesa <rjbuesa <@t> yahoo.com>
To: Victor Wong <vhlwong <@t> yahoo.com>; "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Monday, February 18, 2013 8:42 PM
Subject: Re: [Histonet] Safranin O staining


Believe it or not, not because you are dealing with cells (small as they are) they require longer than 1 hour to be correctly fixed.
This is what I would do: fix the cells for 8 hours minimum → prepare the pellet in agarose → process to paraffin (FFPE) block → section as usual.
Now, check the distaining protocol, consult any histotechnique book because I think 2 min is Weigert is a very short time (remember the mordant) → try different staining times, or follow a known protocol (you can find that in either Peter Gray's or Lillie's books).
René J.

From: Victor Wong <vhlwong <@t> yahoo.com>
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Sunday, February 17, 2013 9:34 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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