In a continuing effort to limit the volume of reagent used in each step of a manual IHC, I have tried TBS and TBST on slides with barriers (expensive) and without barriers. The results were not stellar with the barrier slides - the reagent still escapes. We dewax with hot detergent (may be a contributing factor) and the Tween 20 in the TBST definitely alters the hydrophobicity of the barrier, an effect that is not reversed with a water wash.
Consequently, I have resorted to omitting the buffer wash steps and using distilled water only. The slide surface remains "water repellent" and the added IHC reagents form a pool over the tissue sections. The detection method is a polymer-HRP and there is no increase in background staining in the tissue or on the slide surface. I am assuming that the IHC reagents are prepared in the "optimum" suspension liquid and a buffer is not required. So, I am interested in hearing from anyone why this is a bad idea. Thanks, Tresa [X] [X] _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
