Using a fluorescent microscope is a challenging yet rewarding experience. Knowledge on the proper processing of the samples would greatly help in obtaining optimum results. Fixation, permeabilization and blocking, staining, and mounting are the four general steps when processing a sample using a fluorescent microscope.

In fixation, the goal is to preserve the over-all cellular structure to maintain the original cellular form of the subject on the slide, while preserving the most of the epitopes (the part of a macromolecule that is recognized by the immune system) in question. The time or duration of fixation is also an important factor. If the fixation took too long, epitopes may be damaged. If it is too short, proper preservation of cellular structure may fail. Coagulating fixatives can be employed as preservatives to the samples to be observed in a fluorescent microscope. This includes acetone, ethanol, and methanol. Permeability induction and antigenicity preservation are the benefits of using these solvents. The disadvantage is that they can also cause shrinkage and distortion of tissues.

The next step for processing samples is permeabilization and blocking. There is a need to permeabilize the plasma membrane so that the stain or antibody used can get into the structures in question. It is vital not to destroy the cellular structure of the sample. Blocking would mean using a reagent that will respond with all other potential protein-binding sites and would help in the prevention of any non-specific binding by the antibodies to the sample.

The third step is staining. In order to maximize sample visualization, it is essential to have an optical contrast between its various regions. This is accomplished by staining the sample with a fluorescent label, or a label that either absorbs or reflects light. Fluorescent labels are more flexible and is highly recommended for use with fluorescent microscopes.

Labeling can be of two methods: direct and indirect. Direct labeling involves using a primary antibody, conjugated to fluorescent molecules, specific for the antigen of interest. Indirect labeling involves two steps. First, by means of a primary antibody specific for the antigen of interest, and then a secondary antibody, conjugated to fluorescent molecules, that is specific for the primary antibody. While indirect labeling takes longer, it strengthens the signal which means more fluorescence from the sample. The general protocol for indirect labeling involves: (1) fixation or preservation of the antigen. (2) Block for non-specific interactions using non-immune serum and mild detergent. (3) Adding a primary antibody then washing the blocking solution. A secondary antibody is then added and washed again with buffer.

The last process is the preparation for imaging or mounting. The best way to do it is by mounting the samples under a glass cover slip. Excess moisture from the staining procedure is removed with a Kim wipe, then a small amount of mounting media is put above the sample. There are two methods available to adhere the cover slip to the slide. One is by using clear nail polish and allowing it to dry for 10 minutes. Nail polish can severely damage lenses so it is extremely wise to be cautious. Another method is by using Valap, which is paraffin wax, linseed, and Vaseline in a 1:1:1 ratio. Since it is solid at room temperature, it is heated to liquefy. Valap is good for live cells. If using a cover slip is not possible, try using samples in an aqueous mountant without a cover slip which can then be viewed directly using a water immersion lens.