Signal intensity in tissue sections utilized for fluorescence-based immunohistochemistry can be irreversibly lost by photobleaching, resulting in reduced assay sensitivity, and potentially compromising data. When the molecules in question are of low abundance or when more intense periods of excitation are required, this phenomenon can be extremely frustrating for investigators to overcome. To circumvent the effects of photobleaching, several companies have developed and offer mounting reagents that act to stabilize the activity of fluorophores used in immunohistochemistry. These reagents prolong fluorescence capabilities by chemically inhibiting the generation and diffusion of reactive oxygen species. The Invitrogen Corporation markets the SlowFade Gold® Antifade Reagent which is an improved version of their original SlowFade® Reagent. SlowFade Gold® was designed to increase the photostability of many different fluorophores used when labeling fixed cells and tissues as well as cell-free preparations.
Unlike some other antifade reagents, SlowFade® Gold does not require long curation periods, so samples can be microscopically viewed immediately. The caveat, however, of rapid imaging capabilities lies in the fact that, after mounting with SlowFade® Gold, the fluorescence intensity of the sample will only last for a few weeks. SlowFade® Gold is advertised to minimize fading of multiple commonly utilized dyes, including Alexa Flour dyes, Cascade Blue, tetramethylrhodamine, Texas Red, and fluorescein with relatively little loss of emission intensity. SlowFade® Gold is also available with the blue fluorescent nuclear counterstain, DAPI. It is worth noting here that SlowFade® Gold reagents contain glycerol, and therefore, should not be used for mounting sections that contain lipophilic plasma membrane stains.
The SlowFade® Gold reagent is provided as a premixed and ready-to-use solution. Only 1-2 drops of antifade are required per section. After applying SlowFade® Gold to the stained tissue, the section is carefully coverslipped to avoid trapping of air bubbles. Although the specimen is immediately available for viewing by fluorescence microscopy, the reagent will cure within a couple of hours to overnight, depending on the thickness of the section and the surrounding humidity. The mounted slides can be stored for up to several months without loss of mounting reagent by sealing the edges of the coverslip. The slides can then be stored upright in a covered slide box with desiccant at room temperature, 4°C, or 20°C.
Our lab has recently switched to the SlowFade® Gold Reagent for most of our fluorescent immunohistochemistry analyses. We have been impressed with the reduction in photobleaching, which has traditionally impaired our ability to perform 2- and 3-color immunohistochemistry assays. We have used the reagent to mount sections of small intestinal tissue in which different cell types have been stained simultaneously with Cy-3, Cy-5, and FITC (or Alexa fluor dyes). The resulting images have been utilized for publication. We have found that the fluorescence signal intensity is maintained for up to 4-5 weeks, when the mounted sections are stored at 4°C, or 20°C, and for 2-3 weeks when stored at room temperature.
SlowFade® Gold Antifade Reagent With DAPI From Invitrogen
Greatly extends the photostability of multiple stains commonly used for fluorescence-based immunohistochemistry.
Overall fluorescence intensity and shelf-life of the stained tissue sections is reduced.
The Bottom Line
The use of an antifade mounting reagent is important to successfully perform multi-color fluorescence immunohistochemistry. SlowFade® Gold Reagent does the job well.