AAT Bioquest

How do I prepare fixed cells for the labeling?

Posted August 21, 2019

Preparing fixed cells or tissues for immunofluorescence involves three main steps:
  1. Cell Fixation
  2. Permeabilization
  3. Blocking
Cell Fixation
The purpose of cell fixation is to maintain proteins and cell morphology as much as possible to that of the native state during the processing steps and following imaging. Fixation methods suitable for fluorescence-based imaging and flow cytometry fall into two categories: aldehyde fixatives and alcohol fixatives. Aldehyde-based fixatives, such as formaldehyde, are cross-linking reagents that form intermolecular bridges between various cellular components, typically via free amine groups. Alcohol-based fixatives, which are organic solvents, remove lipids, dehydrates cells and precipitates proteins by reducing their solubility. Of the two categories, aldehyde-fixatives preserve cell morphology much better than organic solvents, but will require a permeabilization step to allow antibodies to enter the cells. Alcohol and acetone based fixatives generally do not require a permeabilization step.
Cell permeabilization can be achieved using mild detergents such as Triton X-100 (0.1 to 0.5%). This detergent will dissolve cellular membranes, without disturbing protein-protein interactions, to allow antibodies to access the interior of cells.
Immunofluorescence staining with antibodies requires a blocking step in order to reduce non-specific binding between the antibodies and molecules in your sample that are not your target. By reducing non-specific binding, it will be easier to identify a positive signal and provide a cleaner end result. Commonly used blocking agents are bovine serum albumin (BSA), casein or gelatin. For primary antibodies make sure to avoid using any serum of the same species as the primary antibody. For secondary antibodies use a heat-inactivated species-specific serum, where the serum species matches the species of the secondary antibody.
Additional resources

iFluor™ Secondary Reagents