I have done extensive IHC work on bone and cartilage samples and in my experience, controlled enzyme digestion such as pepsin at 37dc flat on a slide warmer, or trypsin, or proteinase K are my first choices for AR on bone samples. Complete draining and airdrying is a must before heating the slides, I, like Gayle try to airdry overnight after completely draining the water by standing the slides up for at least an hour. Drying them flat on a slide warmer at 37-40dc is suggested. I do heat them on the same flat slide warmer (mine goes up to only 55dc) to melt the paraffin for maybe 30 min or so.
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 gayle callis Sent: Friday, June 05, 2009 3:28 PM To: 'Troutman, Kenneth A'; 'Histonet' Subject: RE: [Histonet] immunohistochemistry Good advice since actively boiling buffer puts mechanical stresses on section, with bone more easily loosened by this action. It may help to preheat the buffer to 95C before slide/section immersion into retrieval buffer. A friend who is an IHC expert always did this, plus she used plastic coplin jars (safer!). Also, we dry all our decalcified, paraffin embedded bone slides FLAT, at 37C to 40C for overnight, with longer better - be sure slides are well drained before laying them flat. You can go to a 56C paraffin oven for a short time (just enough to melt paraffin) after overnight if you desire, but you find that unnecessary. Gayle M. Callis HTL(ASCP)HT,MT Bozeman MT 59715 -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Troutman, Kenneth A Sent: Friday, June 05, 2009 3:03 PM To: Histonet Subject: Re: [Histonet] immunohistochemistry I would recommend the following: Citrate for 35 min at 95 deg. Cool down for 10 min. (This is our protocol.) Be sure you are using charged slides and I would let them air dry for at least 1 hour before heating and deparaffinizing. That might help, too. Unfortunately, I don't think there is an effective method of retrieival that will completely eliminate the possibilty of floating tissue. Good luck. Ashley Troutman BS, HT(ASCP)QIHC Histopathology Laboratory Department of Pathology Vanderbilt University Medical Center Nashville, TN <http://www.vanderbilthealth.com/main/> <http://www.vanderbilt.edu/> <http://www.mc.vanderbilt.edu/> Date: Thu, 4 Jun 2009 20:01:50 -0700 (PDT) From: Hatem Salim <[email protected]> Subject: [Histonet] immunohistochemistry To: [email protected] Message-ID: <[email protected]> Content-Type: text/plain; charset=iso-8859-1 HI I am Research assistant at Physiology department of Michigan state university. I am using the (ß-Catenin Antibody (Carboxy-terminal Antigen) #9587 for IHC on mice femur sections. I have used this antigen unmasking method: For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0 then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes. The problem is that bone detachment always occurs so I wonder if there is a method of retrieval that prevents bone detachment. > Waiting to hear from you soon. > Thank you for your consideration > Best wishes > Hatem Salim <http://www.vanderbilthealth.com/main/> _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
