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 Health Level 1, Building 15,Princess Alexandra Hospital Ipswich Road,WOOLLOONGABBA Qld4102 Ph: 07 3176 2412 Mob: 0402 139411 Fax: 07 3176 2930 Email: tony_rei...@health.qld.gov.au Web: www.health.qld.gov.au/qhcss/ >>> Victor Wong <vhlw...@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ******************************************************************************** This email, including any attachments sent with it, is confidential and for the sole use of the intended recipient(s). This confidentiality is not waived or lost, if you receive it and you are not the intended recipient(s), or if it is transmitted/received in error. Any unauthorised use, alteration, disclosure, distribution or review of this email is strictly prohibited. The information contained in this email, including any attachment sent with it, may be subject to a statutory duty of confidentiality if it relates to health service matters. If you are not the intended recipient(s), or if you have received this email in error, you are asked to immediately notify the sender by telephone collect on Australia +61 1800 198 175 or by return email. You should also delete this email, and any copies, from your computer system network and destroy any hard copies produced. If not an intended recipient of this email, you must not copy, distribute or take any action(s) that relies on it; any form of disclosure, modification, distribution and/or publication of this email is also prohibited. Although Queensland Health takes all reasonable steps to ensure this email does not contain malicious software, Queensland Health does not accept responsibility for the consequences if any person's computer inadvertently suffers any disruption to services, loss of information, harm or is infected with a virus, other malicious computer programme or code that may occur as a consequence of receiving this email. Unless stated otherwise, this email represents only the views of the sender and not the views of the Queensland Government. ********************************************************************************** _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
