I agree with Tony, the problem is usually underfixation not over with aldehyde fixatives, if you do not stabilize the proteins by fixing (Bryan Hewlett says this takes 24 hrs. no matter the size of the tissue) paraffin processing will adversely affect your tissue and in some cases the proteins of interest will be washed away and lost with no change or no amount of AR to get them back. Researchers are stuck in the days before AR techniques when cross linking of proteins by aldehyde fixation was a problem because we did not know how to retrieve them, but now we do, and the bigger problem is losing antigens from paraffin processing because they have not been adequately fixed to protect them. I recommend 24-72 hour fixation in aldehyde fixative and then the tissues can be placed in 70% alcohol.
Patsy Patsy Ruegg, HT(ASCP)QIHC IHCtech 12635 Montview Blvd. Ste.215 Aurora, CO 80045 720-859-4060 fax 720-859-4110 www.ihctech.net www.ihcrg.org -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Tony Henwood Sent: Thursday, June 04, 2009 5:22 PM To: [email protected] Subject: RE: [Histonet] Storage of tissues in PFA before dehydrationandembedding Nick, Well after 30 years of doing ICCs, the worst results are nearly always with tissues that have not been adequately fixed in formalin. This is based on using over 300 different antibodies in both adult and pediatric settings. The following paper shows an example of what can go wrong with under-fixed tissue: Gomes L, Mackie N, Catchpoole D, Henwood T (2008) "Test Your Knowledge" J Histotechnol 31(4):138-184 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: Nicholas David Evans [mailto:[email protected]] Sent: Friday, 5 June 2009 8:56 AM To: Tony Henwood Subject: RE: [Histonet] Storage of tissues in PFA before dehydration andembedding Thanks for the helpful response. After a consensus of views I decided to store at 70% ethanol for several days following 3-4hrs in 4% PFA. I think many people use paraffin embedded tissues for ICC and over-fixation is deleterious for this. 4% Paraformaldehyde (PFA) = 4g PFA dissolved in 100 mL PBS. Indeed when in solution it is present as formaldehyde. I will consider your suggestion of frozen sections if I have poor results. Thanks again for the e mail and best wishes Nick -----Original Message----- From: Tony Henwood [mailto:[email protected]] Sent: 03 June 2009 16:54 To: Nicholas David Evans; [email protected] Subject: RE: [Histonet] Storage of tissues in PFA before dehydration andembedding Nick, Where did you hear that "storage in PFA for longer than 24 hrs may adversely affect my tissues"? The beauty of 4% formaldehyde (especially if it is buffered) is that it does not adversely affect tissues, in fact it protects them brilliantly from the rigours of subsequent processing. If it is immunohistochemistry using formalin-labile antigens that you are worried about then use frozen sections not FFPE sections. The major cause of poor morphology in FFPE tissues is inadequate formalin fixation. If you want ethanol fixed tissues (and rotten morphology) then leave out the formalin fixation step altogether. AND how do you fix it in 4% polyformaldehyde? Surely when it is in solution it becomes formaldehyde? 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: [email protected] [mailto:[email protected]] On Behalf Of Nicholas David Evans Sent: Thursday, 4 June 2009 3:59 AM To: [email protected] Subject: [Histonet] Storage of tissues in PFA before dehydration andembedding Dear all, I am doing some experiments where mouse skin tissue is harvested for immuno and in situ staining at 2, 4, 6 and 8 days following a treatment. The usual protocol used in our lab (for other tissues, usually bone) is to fix overnight in 4% PFA before dehydration and paraffin embedding. As I value my weekends, I would obviously prefer to do the dehydration (which we do in ethanol series, an hour in each of 30, 50, 70, 90, 95, 100% ethanol = 6 hours, before overnight in 100%) and embedding of all samples on the same day. I understand that storage in PFA for longer than 24 hrs may adversely affect my tissues. Is it possible to store for longer in a lower concentration of PFA, or in another buffer until I am ready to dehydrate and embed? In short, I would appreciate if anyone could suggest a way to plan this experiment in the most efficient way. With thanks and best wishes Nick Evans Dept Surgery Stanford University _______________________________________________ Histonet mailing list [email protected] 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. ********************************************************************** ********************************************************************* 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 [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
