[Histonet] Re: Histonet Digest, Vol 5, Issue 29

JColCLEFA <@t> aol.com JColCLEFA <@t> aol.com
Mon Apr 19 20:53:30 CDT 2004

Our protocol for TDT is Citrate buffer pH 6.0 @60c for 25 minutes in a water 
bath, H2O2 block, caseinate block, cell marque polyclonal tdt concentrate at a 
1:50 dilution, 30 minutes RT primary antibody, and we use a combined control 
of tonsil, thymus and lymphoblastic lymphoma.(the tonsil is included to make 
sure we aren't getting false positives and should NOT stain. ) We use goat   
biotinylated link, (streptavidin biotin secondary reagent kit,) hrp label, DAB 
chromogen. No protein digestion. In working up the antibody , we found staining 
of negative cases was our biggest problem , and was caused by routine heat 
induced epitope retrieval methods. (25 min in citrate 6.0 @95C and EDTA pH 8.0 
15- 25 min both caused negative cases and normal tonsil to stain. ) Questions? 
just ask
J. Coleman QIHC

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