Emily, Unfortunately, there are a lot of superfluous steps involved in ISH. It reminds me of the initial IPX protocols. There were many unfathomable steps included that seemed to incite a feeling of magic required to obtain results. Simplifying the technique was one of the main reasons why routine labs were able to utilise it. I hope the same will occur with ISH.
IPX and ISH are similar: Block the endogenous enzyme Reverse the formalin cross-linking Apply the probe or antibody Demonstrate the bound probe or antibody. Stringency washes might be required if there is a high possibility of cross-hybridisation. Tissue sections will react differently to Molecular biology nucleic acid containing tubes and gels Granted, for DNA hybridisations you will need to denature the DNA strand but overall that is about it. I would keep it simple and go from there. Many of the added steps were added by researchers because they seemed like the right thing to do not because they were required. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Emily Sours Sent: Thursday, 20 August 2009 2:49 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] in situ with paraffin sections Hello I've been reading a few protocols for RNA in situ hybridization with paraffin sections. One of the suggestions is to dehydrate the sections on the first day after rehydration and pre-hybridization processing (para fix, proteinase K, acetylation, pbs washes between all). This seems counter intuitive to me, or at least unnecessary. Any suggestions why this would be done? Our protocol does not involve this, by the way, and it works fine except for the tissue tearing. Emily "One of the defining characteristics of modern surgery was that patients ought to survive it." --Peter Stanley, For Fear of Pain: British Surgery, 1790-1850 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ********************************************************************* This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. ********************************************************************** _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet