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 <vhlw...@yahoo.com>
To: "histonet@lists.utsouthwestern.edu" <histonet@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
_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Reply via email to