It is advantageous for one to perform an optimal number of experiments at a given time using the most cost-effective strategies. Experiments that involve immunohistochemical techniques on tissue samples are some of the most costly to perform. In particular, sera, as well as primary and secondary antibodies, are expensive reagents for applications such as immunofluorescent and immunoperoxidase (DAB) labeling. The Liquid Blocker Super PAP Pen (Product# 71310) from EM Sciences solves many of these issues and outperforms the rest by allowing for flexibility within experiments.
Several companies offer antibodies that must be applied at high concentrations for proper labeling. When applying antibody solutions to a tissue section as large as one inch in diameter, as in the case of human brain samples, several hundred microliters of reagent are required to ensure complete coverage. It is also necessary to make certain there is enough reagent to allow for spillover onto areas of the slide where there is no tissue. This can result in the use of almost all the antibody product, costing hundreds of dollars for a few slides in a given experiment.
The PAP pen solves this issue by allowing one to draw a hydrophobic barrier around the tissue. Consequently, solutions remain within the boundary marked by the pen and a significant amount of reagent can be saved for other experiments. This is especially valuable in our lab when using mouse samples. Without a PAP pen, we must apply 400 – 500ul of sample in order to cover the slide. In contrast, with the PAP pen only about 100ul of reagent are required per sample.
Another cost-effective strategy is to mount at least two sections on one microscope slide. However, this strategy in itself can be expensive if only one sample is required for a given condition. This results in either the waste of one tissue sample, or if one decides use the second section as a duplicate (even when it is not necessary), there is need for additional reagents to cover both sections on the slide. The PAP pen can also solve this issue by allowing one to draw a barrier separating the two samples. One can then apply two different antibodies of interest without the worry of spillover to the adjacent sample.
The PAP pen can be used for both paraffin and frozen samples in the same manner. The dehydration, antigen retrieval, permeabilization and washing steps are completed as described in standard protocols for immunodetection via fluorophore or peroxidase on paraffin and frozen tissue. Prior to blocking, I then remove the slides one at a time from the wash buffer, pat them dry on an absorbent paper towel, and draw an outline around the sample with the pen. I allow the marking to dry for a few seconds, and immediately apply block buffer to keep the tissue from drying out. The product insert includes slightly different directions calling for drawing the outline and then washing for 5 minutes in wash buffer. This method also proves to be exceptional.
The PAP pen is shipped in a dry state. To start the flow of the hydrophobic solution, the tip of the pen is depressed several times on a paper towel until the tip is completely saturated with solution. Care is required during application to the slide. One must ensure not to depress the pen on the slide or it will leak and destroy the tissue. It is also advised not to make a barrier too close to the tissue or it can dry out the edges of the samples. I generally only draw a line directly in the center of the slide to separate the two samples and then mark a line near the edge of the slide to form a barrier. The slides we generally use are 1 in x 3 in and tissue samples are no larger than 1 inch in diameter. I generally draw the outline at least 5 mm away from all edges of tissue.
The Liquid Blocker Super PAP Pen is insoluble in alcohols and acetone and can withstand temperatures up to 120°F. It does not interfere with aqueous mounting media for immunofluorescence and can also be removed with xylene prior to coverslipping for light microscopy techniques. These are the key features that make this PAP pen superior to others. EM Sciences also offers a large as well as small tip version of this product. We prefer the large tip to ensure a thick barrier between samples. The Liquid Blocker Super PAP Pen is a worthwhile investment, as it will provide one with some flexibility in designing immunostaining experiments.
Surita Banwait
Research Associate II
Buck Institute for Age Research
Morphology & Imaging Core