[Histonet] Safranin O staining

[Victor Wong]

Dear Tony,
 
Thanks for your prompt reply.
 
I fix the cells in 10% NBF for 1 hour, then changed to PBS and keep at 4C fridge.  To prepare cell block, I use a heating water bath and I usually need to set the heater to higher than 150C to liquidify the agarose (2% in PBS) without boiling.  The agar did not melt when heated at lower temperature.  I do think the agar is at less than hundred degree C but may be at 70C.  After then, I trimmed the block and fix again in formalin before putting in 70% alcohol.  Any comments?
 
This is the first time I am working on cell block.  May I have your processing protocol?
 
Best Regards,
Victor
 

From: Tony Reilly <Tony_Reilly <@t> health.qld.gov.au>
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>; Victor Wong <vhlwong <@t> yahoo.com> 
Sent: Monday, February 18, 2013 1:50 PM
Subject: Re: [Histonet] Safranin O staining


Hi Victor

I have been using agar cell blocks for over 30 years.  It has been my experience that it is better to fix the cells in formalin prior to embedding in the agar to prevent damage to the cells from the heat of the agar.  Another important step is to gently heat the agar so that it is just above melting point to also minimise the heat affect.  If you are using microwaves to melt your agar do the following.
-calibrate your microwave so that the temperature achieved is minimal
-do not set and walk away, watch and stop heating as soon as melting is achieved
-aliquot agar into small batches as the microwaves affect the agar such that the melting point increases with each use elevating the temperature of the agar.

regards
Tony





Tony Reilly  B.App.Sc. , M.Sc.
Chief Scientist, Anatomical Pathology
Pathology Queensland-PA Laboratory 
________________________________________________
Health Services Support Agency | Department of HealthLevel 1, Building 15,Princess Alexandra Hospital
 
Ipswich Road,WOOLLOONGABBA  Qld4102
Ph: 07 3176 2412
Mob: 0402 139411
Fax: 07 3176 2930
Email: tony_reilly <@t> health.qld.gov.au
Web:  www.health.qld.gov.au/qhcss/
 
 

>>> Victor Wong <vhlwong <@t> yahoo.com> 2/18/2013 12:34 pm >>>
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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