[Histonet] IHC wash with dH20 / follow-up

[Goins, Tresa]

Thanks for the responses to my question re IHC wash without buffer.  It made me examine the question more thoroughly with empirical comparisons.

The Dako IHC manual is a good reference.  But, the take home message I got after reviewing it didn't alter my view that buffer wash may not be required.  Following the initial electrostatic interaction that occurs between antibody and antigen in the milieu of the antibody diluent, the Ab-Ag bond is maintained via Van der Waals and hydrophobic forces.  No matter what the slide is rinsed with, I don't think this microenvironment is going to be altered.

In our hands, IHC results with twenty antibodies has not been adversly effected by using dH2O in place of wash buffer (TBST in our case) - staining intensity and contrast with the water wash is equivalent to buffer washed slides.

I located a reference that supports the use of a water wash (Hallelujah - I'm not crazy) in:

      TECHNICAL IMMUNOHISTOCHEMISTRY:Achieving Reliability and Reproducibility of Immunostains.

      Rodney T. Miller, M.D.
      Director of Immunohistochemistry
      ProPath Laboratory, Inc.
      8267 Elmbrook, Suite 100, Dallas, Texas 75247-4009
      Phone (214) 237-1631 FAX (214) 237-1770
      www.propathlab.com E mail: rmiller <@t> propathlab.com<mailto:rmiller <@t> propathlab.com>

            An excerpt from the reference states:

            Although previously I never thought of using anything else for rinsing steps, a conversation with another immunohistochemist (Dave Tacha, BioCare, Walnut Creek, CA) led me to try distilled water with Tween 20 (DW/Tween20) rather than PBS for the rinsing steps.  When we evaluated the performance of DW/Tween 20 for our rinsing solution between steps, it performed just as well as using PBS with Tween 20, so we now routinely use DW with 0.2% Tween 20 (2 ml Tween 20 in 10 liters of DW) for all of our rinses that previously used buffer.  This has saved us substantial amounts of reagent cost as well as reagent preparation time, since preparing DW/Tween 20 is far easier and cheaper than preparing our previous PBS buffer rinsing solutions.

OK, detergent is still present, but with polymner-based IHC, background has not been a problem . . . . .yet.
At the very lease, the cost of Tween 20 and distilled water is a whole lot cheaper than Tween 20 and buffer.

Happy validating!

Tresa




Tresa Goins
Histopathology Section
Montana Veterinary Diagnostic Lab
Bozeman, MT 59715
406-994-6353