What Is a Pap Pen?
A pap pen is used to create a water-repellent barrier around a sample on a glass slide, primarily for immunohistochemical staining and other biochemical experiments involving fluorescent antibody reactions.
Immunohistochemical staining is a biochemical technique that utilizes antibodies to identify the location of proteins and other antigens in tissue sections. After the antigen is labeled with an antibody, the antigen-antibody complex is visualized using fluorescence detection or other methods. The pap pen is used to draw a hydrophobic circle around the specimen on a glass slide, which helps prevent the loss of antibodies and specimens during the process. It is sometimes referred to as a PAP pen, named after the peroxidase anti-peroxidase (PAP) method, a common technique in fluorescent antibody staining.
Uses of a Pap Pen
Pap pens are primarily utilized in biochemical experiments such as immunohistochemical staining and in situ hybridization on glass slides under a microscope.
By drawing a water-repellent barrier around the specimen, the pen helps confine the specimen and antibodies on the glass slide, ensuring they do not wash away during processing. Although referred to as a “pen,” it is not designed for general writing but specifically for laboratory applications.
Principle of a Pap Pen
Immunohistochemical Staining
Immunohistochemical staining is a technique that specifically detects antigens in cells and tissues using antigen-antibody reactions. This method can employ fluorescent or enzymatic labeling of antibodies. The key steps include:
- Specimen preparation
- Antigen activation
- Antibody application
- Antibody detection
During specimen preparation, the pap pen is used to draw a water-repellent circle that helps contain the sample and reagents on the glass slide, preventing them from leaking during subsequent steps.
Both direct and indirect antibody labeling methods are utilized. In direct labeling, primary antibodies are directly tagged with enzymes or fluorescent probes. Indirect labeling involves a primary antibody and a secondary antibody, which specifically binds to the primary antibody and is labeled with an enzyme or fluorescent probe. Detection is achieved through colorimetric (enzyme-antibody method) or fluorescence-based methods (fluorescent antibody method).
Functions and Use of a Pap Pen
The pap pen is designed to draw repellent lines on glass slides according to the size, shape, and number of samples. Its ink is water-repellent, insoluble in alcohol and acetone, but soluble in xylene, allowing for easy removal. Most pap pens are heat-resistant up to 120°C (248°F).
To use, activate the pen by pressing the tip against a hard surface to release any trapped gas. Shake well, then press the tip on a microscope slide repeatedly until the ink flows. Draw lines around each sample, and store the pen cap-side down at room temperature after use. If excess ink flows, remove it by dabbing on an unwanted piece of paper.
Types of Pap Pens
Pap pens are available from several manufacturers and come in various line thicknesses including 2 mm, 3 mm, and 4 mm. Ink capacities range from 2.5 mL for the fine line type to 5 mL for the thick line type, with the fine line type usable approximately 500 times and the thick line type about 1000 times. Common ink colors are light green and light blue.
While pap pens generally require a surfactant-free surface, some are formulated to remain stable in buffers containing surfactants such as Tween 20 or Triton X-100.