Preparing Fixed Cells for Labeling

Fixing and permeabilizing cells generally locks them in place and makes it possible for larger molecules such as antibodies to access the interior of the cell for better targeting of the protein or condition you're interested in. But, fixed and permeabilized cells are dead, and you lose the ability to look at dynamic biological processes.

The information below will help you decide whether fixation and permeabilization are the right treatments for your investigation.

Figure 1. Formaldehyde fixation essentially locks cellular structures in place.

Fluorescent stains vary in their ability to keep producing a signal after a cell has been fixed. With some stains, you can label cells while they are alive and then fix them without a loss in signal. The more common approach, however, is to fix, permeabilize, and block your cells and then stain them with fluorescent dyes and/or antibody conjugates.

Formaldehyde is the most commonly used fixative; it works by chemically bonding adjacent macromolecules, such as proteins, together. This process is known as crosslinking. Most available formaldehyde preparations are actually paraformaldehyde (PFA, polymeric formaldehyde) dissolved in water or a buffer. The free methanediols in the resulting solution are reactive with amine groups on proteins and other cellular structures that contain nitrogen. PFA also solubilizes some lipids in cellular membranes. PFA is commonly diluted to 3.7–5% v/v and is applied to cells for 10–15 minutes.

The permeabilization step removes more cellular membrane lipids to allow large molecules like antibodies to get inside the cell. Thermo Scientific™ Triton™ X-100 and NP-40 are detergents commonly used at 0.1–0.5% (v/v, in PBS) for permeabilization. A permeabilization time of 10–15 minutes is a good starting point, but if that isn’t working well for your target you might need to try a shorter time or a different detergent. These detergents will also permeabilize the nuclear membrane, so they are suitable for a variety of target locations.

Figure 2. The removal of cellular membrane lipids in the permeabilization step allows large molecules access to the interior of the cell.

Figure 3. The use of protein-based blocking agents reduces nonspecific staining.

If you are going to use antibodies to label structures in your fixed and permeabilized cells, using a blocking solution before your primary antibody step can be helpful. Blocking is usually performed with a solution containing an excess of protein that serves to reduce the amount of nonspecific binding in your sample. This can be important if your primary or secondary antibody has a tendency to interact with molecules in the sample that are not your target. Reduction of nonspecific “background” staining, most likely due to hydrophobic interactions between the antibody and non-target molecules, will make it easier for you to identify a positive signal and will give you a cleaner end result.

The most common types of blocking solutions for ICC are 3% (w/v) bovine serum albumin in PBS and/or a 10% (v/v) solution of heat-inactivated species-specific serum in PBS, where the serum species matches the species of the secondary antibody. For example, if you are using a mouse anti–rabbit IgG secondary antibody, chose normal heat-inactivated mouse serum to make your blocking reagent.