Several messages asked about the volume of antibodies/immunoreagents used on
sections. Some of those questions were specifically targeted towards the
DAKO Autostainer. Since these questions/responses indicate that some of us
on Histonet are not doing IHCH correctly, I feel compelled to highlight a
few seemingly trivial/minuscule but crucial details.

First of all there is not and can not be a general rule about the volume of
reagents applied to a section (manually or by a stainer). This volume,
however, is determined basically by two factors: 1./ the size of the area
occupied by sections; and 2./ the duration and temperature of incubation.

Pro primo: 
It is important to cover the tissues fully, to have uniform exposure and to
avoid any edge effects (created by evaporation of reagents at the edge of
the drop) which could result in false-positive areas. Therefore if one has
just a small piece of tissue (such as a biopsy sample), less reagent volume
is needed. Similarly, for large tissue sections or for multi-tissue
sections, which occupy almost completely the whole surface of the slide, a
much larger volume is needed. The rule of thumb in my lab is that for about
1 square inch of tissue to be covered, we apply 400 microliter. Also our
buffers always contain Tween which influences the spreading characteristics,
for other buffers this number could be different. Also please realize that
this volume is not based on scientific calculations, but a result of a
practical compromise of not using/wasting too much antibody and money while
still providing uniform exposure of the samples to the reagents.

Pro secundo:
Evaporation is lower at 4oC, therefore a smaller volume can be used.
Moreover, the longer the incubation the more antibody is needed to avoid
evaporation.

Pro tertio: 
Additional comments: PAP pen border could reduce the volume of antibody
needed. In the DAKO stainer reagents are applied in three different spots.
Prudent and considerate planning during sectioning could reduce the volume
applied to the section significantly (resulting in huge savings on a long
run). Also by running a PAP border along the long edges of the slide, an
even smaller volume would suffice.

I'll be glad to continue this discussion on- or off-line, if anybody has
further questions or comments.

Laszlo Komuves

Correspondence: 
László G. Kömüves, Ph.D.
Senior Scientist, Histology
COR Therapeutics, Inc.
256 East Grand Avenue
South San Francisco CA 94080
Phone: (650) 244-6855,  Fax: (650) 244-9270