I work with bone samples trimmed to approx 35mmx35mmx4mm. Our general procedure is:
Fixation: untrimmed samples stored in 10% NBF @4°C until trimming. Slices placed in fresh 10% NBF for at least 12h. Decalcification Benchtop - 10% formic acid for 10 days (change soln every 3 days), rinse for minimum 1-2 hours. Then 10% EDTA solution for 2-3 weeks (change weekly). The time in formic acid is limited at 10 days to prevent excess damage to tissue/loss of nuclear staining. The EDTA is a chelating agent and is slower at decalcifying tissue but does little damage. Microwave processor - 3 days in 10% formic acid @35°C, rinse as above, then 3 days in 10% EDTA @35°C. For both methods I recommend placing a layer of empty cassettes at the bottom of you container to allow solution to circulate under samples. After the EDTA step, it is VITAL that samples are rinsed for at least 3 hours to remove any remainng salts. These precipitate during processing and make sectioning very difficult. You may need to leave samples longer in EDTA depending on their size. There are various methods in text books for determining decalcification end point. We have a MicroCT scanner so just do a quick xray scan to check our samples. Processing. We have a Sakura Tissue Tek 5. Samples processed for 5 hours each through 50%, 70%, 90% and 3x100% Ethanol/IMS. Then 4 hours x3 changes for chloroform, then 2 hours x4 changes of wax. Sectioning Make sure block are well chilled. Some samples may need soaking in water to get good sections. We trim in samples then place face down on a melting ice block whilst trimming in the other samples. This means our samples soak and chill at the same time. If you get small areas of mineral left, you can perform surface decal with 10% fromic acid. Hope this points you in the right direction. If you can, try and go to this years NSH convention. Lots of very helpful people there, especially the Hard Tissue Committee. Best of luck. Andrew Prior Histologist Smith &Nephew Research Centre York Science Park UK andrew.pr...@smith-nephew.com >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> ------------------------------ Message: 21 Date: Wed, 24 Feb 2010 17:34:06 -0800 (PST) From: Victor Wong <vhlw...@yahoo.com> Subject: [Histonet] Decalcification and processing of large bone To: Histonet@lists.utsouthwestern.edu Message-ID: <347173.34928...@web52706.mail.re2.yahoo.com> Content-Type: text/plain; charset=iso-8859-1 Dear all, I am going to decal and process PIG femur and lumbar vertebrates. I only have experience on soft tissues. Anyone can suggest any protocols in decalcification and processing? Many thanks in advance. Cheers, Victor >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Confidentiality. This electronic transmission is strictly confidential to Smith & Nephew and intended solely for the addressee. It may contain information which is covered by legal, professional or other privilege. If you are not the intended addressee, or someone authorised by the intended addressee to receive transmissions on behalf of the addressee, you must not retain, disclose in any form, copy or take any action in reliance on this transmission. If you have received this transmission in error, please notify the sender as soon as possible and destroy this message. Smith & Nephew UK Registered in England and Wales No.4421171 with registered office at 15 Adam Street, London WC2N 6LA _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
