Fixation and Permeabilization in ICC IF

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What is the significance of fixation?

Fixatives preserve cellular morphology, integrity and structure by preventing proteolytic enzyme induced autolysis of cells and the process of putrefaction (cellular decay). Fixatives also enhance the rigidity and mechanical strength of cells, which is critical to withstanding the various, sometimes rigorous, steps of the immunostaining procedure. Cells should be fixed immediately following removal from cell culture conditions to limit autolysis and putrefaction. If there is a delay in fixation or if fixation is incomplete, the antigen may disperse into neighboring sub-cellular regions. This results in misleading staining as shown in the example below.

Poor fixation Optimal fixation
Poor fixation in ICC/IF Optimial fixation in ICC/IF
GM130/Golga2, a Golgi marker, is detected in HeLa cells under different fixation conditions using 5µg/ml of GM130 antibody [NBP2-53420] and DyLightTM 488 conjugated secondary antibody. Cells were counterstained with phalloidin (cytoskeleton, red) and DAPI (nuclei, blue). The ICC/IF image on the left shows improperly fixed cells and Golga2 seems to have dispersed into the nucleus and cytoplasm. The right ICC/IF image shows a properly fixed sample with Golga2 localized to the Golgi apparatus.

Major type of fixatives

Cross-linking fixatives

Aldehydes, such as formalin or formaldehyde, are the preferred fixative for preserving cell morphology and are well suited for immunostaining of membrane proteins. These fixatives crosslink proteins via free amine groups, forming intermolecular bridges and a network of linked antigenic proteins. Crosslinking masks antigens and reduces target antigenicity which may be perpetuated by a long fixation time. Cells are typically incubated with 4% formaldehyde or 10% formalin solution for 10-20 minutes at room temperature.

Precipitating fixatives

Some organic solvents, such as methanol, acetone, and picric acid, act as strong dehydrants and cause the precipitation of cellular proteins. While these fixatives are effective at preserving cellular architecture, they can remove small soluble molecules and lipids. In addition, precipitating fixatives are not recommended for use with overexpressed fluorescent proteins (e.g. GFP) because they can denature these proteins. In a standard fixation protocol, ice-cold methanol is added to cells for 10-20 minutes at 4˚C. Other protocols may involve acetone, a milder fixative compared to methanol or a mixture with an equal ratio of chilled methanol and acetone (1:1). Cells fixed with either methanol or acetone may not require a permeabilization step.

Is there a difference between 10% Formalin and 4% Paraformaldehyde (PFA)?

Paraformaldehyde (PFA) is a powder form of polymerized formaldehyde that needs to be dissolved in PBS before it is used as a fixative. Formalin is a commercially available, saturated formaldehyde solution (37% w/v) that also contains methanol as a stabilizer to prevent the polymerization of formaldehyde. A 3.7% PFA solution is equivalent to a 10% formalin solution.

What are the major advantages and disadvantages of various fixatives?

Fixative Major advantages Major disadvantages*
Formaldehyde Universal fixative, recommended for testing novel antibodies or antigens. Ideal for preserving morphology and staining membrane proteins. Some antibodies may not detect formaldehyde fixed antigens. Excessive crosslinking of antigens may reduce the signal.
Methanol Good choice for aldehyde sensitive epitopes. Additional permeabilization is not required. Some antibodies may not detect methanol fixed antigens. Highly volatile and flammable. Not good for over-expressed fluorescent probes.
Acetone Good choice for aldehyde or methanol sensitive epitopes. Additional permeabilization is not required. Less harsh than methanol. Some antibodies may not detect acetone fixed antigens. Highly volatile and flammable. Not good for over-expressed fluorescent probes.

* Always check the application notes and the customized protocols, if available, on the datasheet of antibodies for these limitations.

What is cell permeabilization and why is it important in ICC/IF?

Under normal conditions, antibody molecules are too large and ionic to pass through the cellular membrane to detect intracellular proteins. Permeabilizing the cells through methanol or acetone fixation, or with the use of a detergent, allows antibodies to pass through the cellular membrane and enter the cell. The most common reagent used for cell permeabilization is non-ionic detergent, Triton X-100. Other milder permeabilizing agents include digitonin or related saponin compounds.

Permeabilization Agent Working Concentration and Incubation Time Major Advantages Major Disadvantages
Triton X-100 0.1% -0.4% in PBS, 10-15 minutes Most common, permeabilize all lipid bilayers including the nuclear membrane. High concentrations or longer incubation may lyse cells. Non-selective.
Saponins/Digitonin 0.1 % in PBS, 5-7 minutes Reversible. Maintains the integrity of protein surface antigens. Common alternative for cells sensitive to Triton X-100. Doesn’t permeabilize the nuclear membrane.
Methanol 100%, 10 minutes Dual agent acting as a fixative and permeablization agent. Recommended for phosphorylated and nuclear antigens. Can damage the cell membrane, the microtubules and other organelles.