Preparation of cells for fluorescence microscopy

There are different steps to take into account when preparing cells for fluorescence microscopy:

• Fixation of cells: the aim is to "freeze time" for the cells and maintain their cellular structure in order to visualize it under the microscope. However, when performing live-cell imaging this step is not needed. Fixation can be carried out by using two types of chemicals:

  • Aldehydes: They cross-link proteins to preserve their structure. The most used ones are Formaldehyde, Paraformaldehyde, or Formalin. The use of aldehydes to fix cells normally requires a quenching step to reduce the autofluorescence generated by some by-products created when they react with amines and proteins.

  • Alcohols: They precipitate proteins to maintain their structure. The most common one is methanol.

• Permeabilization: the goal for this step is to provide access to antibodies and/or chemical probes inside the cell, if fixation with aldehydes was used before. Due to the nature of the cell membrane, detergents are the perfect reagents to permeabilize the cells, Triton X-100 being the most common. Users should evaluate if this step is needed depending on the fixation used and the cellular location of the molecule of interest.

• Blocking: this step is needed if antibodies are used, as it reduces the non-specific binding of antibodies to sites other than the target antigen. Blocking agents are normally protein solutions that compete for non-specific binding sites before antibodies are added. Bovine serum albumin (BSA), a solution of non-fat dry milk, gelatin, or Tween 20 (a non-ionic detergent) are commonly used blocking agents.