The ImmEdge Hydrophobic Pen from Vector Laboratories is a very useful tool for those performing immunohistochemistry utilizing paraffin-embedded tissue sections. Previously we were using plastic coverslips, which would sometimes introduce unwanted air bubbles and occasionally interfere with the tissue section itself. In our hands, the ImmEdge pen is simple to use and one of its main advantages is that significantly less antibody need be applied. In general, we use 50 – 100µl of antibody per section (depending on section size) when using the ImmEdge pen compared to 100 – 200µl of antibody when using the plastic coverslip method.
The basic idea of the ImmEdge pen lies in the hydrophobic characteristics of its contents. This enables the user to circumscribe around the tissue section in a defined manner, forming a hydrophobic barrier. Upon application of the antibody the solution remains precisely on the tissue section within the boundaries of the hydrophobic barrier.
The pens are supplied in packs of two and when received are in a dry state. To start the flow of the hydrophobic solution, the tip of the pen is depressed several times until the tip is completely saturated with solution. It is advisable not to be too vigorous in this procedure as this can lead to subsequent pen leakage. When using the pen, care should be taken that the hydrophobic barrier is completely dry before applying the antibody solution and it should be ensured that no gaps are present. If the flow of solution becomes inadequate, the tip of the pen can be depressed to increase the flow.
The hydrophobic barrier solution is insoluble in most alcohols and acetone so once applied it is often unnecessary to reapply at subsequent steps of standard immunostaining protocols. For those dehydrating sections following processing of the sections for visualization via light microscopy the barrier is conveniently removed upon immersion in either xylene or commonly-used xylene substitutes. In our extensive experience the presence of the barrier does not significantly interfere with routine immunofluorescence protocols when using aqueous mounting medium such as Vector Laboratories VectaShield.
Jonathan M. Shillingford, Ph.D.
Research Associate
Department of Cell Biology
Lerner Research Institute