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