Aldehyde Fixation

Aldehyde Fixation

Aldehyde fixation is a technique used in the preparation of biological samples for immunohistochemistry and related staining methods. Its purpose is to preserve tissue integrity by halting degenerative processes, such as autolysis, which can rapidly compromise sample quality. The fixation method relies on the chemical reactivity of aldehydes, which stabilise proteins through the formation of cross-links. However, this can inadvertently mask antigenic sites, affecting the intensity and specificity of immunological staining.

The choice of fixative, duration of fixation, and subsequent antigen retrieval procedures are all critical factors to consider when designing protocols for reliable and reproducible immunostaining. These factors can vary significantly between different antibodies, and it is important for end users to consider optimizing each antibody separately prior to staining.

Purpose of Fixation

Once tissue is separated from its blood supply or cells removed from their growth media, several degenerative processes commence. Processes like putrefaction (degradation due to the presence of microbes) or autolysis (protein digestion from the release of intracellular enzymes by the rupture of organelle membranes).

Fixation attempts to arrest or prevent these processes and preserve the sample for study and is commonly incorporated into immunohistochemistry, immunocytochemistry, immunofluorescence, and flow cytometry protocols.

Protein Cross-Linking and Antigen/Epitope Masking

Aldehydes react with amine side chains to form Schiff bases which, in turn, react with proteins and peptides to form stable methylene bridges (see Fig. 1). As a result, amino acid sequences that are recognized by antibodies in unfixed tissue may become hidden or ‘masked’ behind the induced protein folding caused by these cross-links. This reduces antigen accessibility and consequently decreases staining intensity. The extent of ‘masking’ also depends on the antigen being studied, some are more susceptible to aldehyde than others.

Fig. 1. Steps in the formation of protein crosslinks. 1) An aldehyde (R–CHO) adds to a nucleophilic amino group—typically the ε-amine of a lysine residue or an N-terminal α-amine—to give a hydroxyalkyl adduct (or a methylol adduct for formaldehyde, when R = H). 2) Loss of water results in a Schiff base, which at physiological pH is in equilibrium with its protonated, strongly electrophilic iminium form. Steps 1 and 2 are both readily reversible. 3) A nucleophilic group from the same or a different protein reacts with the Schiff base to form a covalent cross-link. These cross-links may reduce epitope accessibility and thereby contribute to epitope masking.

Considerations When Immunologically Staining Aldehyde-Fixed Tissue

Three key considerations for staining aldehyde-fixed tissue:

1. Type of fixative—below are a few examples of fixatives used in tissue preparation. However, it is good to keep in mind that other aldehyde-based fixatives (glutaraldehyde, glyoxal, acrolein, etc) are used in laboratories:

a. 10% neutral buffered formalin (NBF)—10% formalin is a 1:10 dilution of a saturated solution of formaldehyde in water. Because the maximum concentration of formaldehyde in water is 40%, a 1:10 dilution of a saturated solution of formaldehyde in water would contain 4% formaldehyde. Methanol is typically added to saturated solutions of formaldehyde to inhibit polymerization.

b. 4% paraformaldehyde (PFA)—paraformaldehyde is polymerized formaldehyde. Once mixed with water and heated, it depolymerizes and becomes formaldehyde. In terms of formaldehyde concentration, 4% PFA is equivalent to 10% NBF. However, since PFA solutions need to be kept refrigerated to inhibit re-polymerization, tissue penetration is slower than NBF and fixation may take longer.

c. Periodate-lysine-paraformaldehyde (PLP)—an alternative fixative first published by McLean and Nakane (1974) for the preservation of carbohydrate-rich/glycosylated cell surface antigens. Periodate oxidizes carbohydrate residues to create aldehyde groups. Paraformaldehyde reacts with the aldehyde groups on the carbohydrates and the lysine residues, creating cross-links that stabilize the tissue. Lysine cross-links periodate-oxidized molecules to other proteins, further stabilizing the tissue structure. PLP is considered a milder fixative that results in reduced levels of antigen/epitope masking and, thus, reduces the need for downstream processes like antigen/epitope retrieval.

Table 1. Fixative comparison.

2. Fixation time.

The longer a sample is exposed to an aldehyde fixative, the greater the opportunity for more methylene bridges/cross-links to form. This increases the probability that any epitope becomes ‘masked’ and unavailable for antibody binding, leading to a decrease in staining intensity (Pollard et al. 1987). Most published guidelines recommend 24 h to 36 h fixation periods (Engel and Moore 2011).

3. Antigen/epitope retrieval.

Antigen retrieval attempts to restore antigenicity by breaking the cross-links and re-exposing the epitopes. This is commonly achieved by a combination of physical (heating) and chemical (pH) manipulation or through enzymatic (protease) methods. These processes are, by their nature, destructive, and care and attention must be taken to ensure that the tissue is not exposed to too much damage, making it unsuitable for staining.

Example IHC Images

Fig. 2. Staining of formalin-fixed, paraffin-embedded human tonsil. Stained with Mouse Anti-Human CD68 antibody (MCA5709), following heat-mediated antigen retrieval using citrate buffer (pH 6.0). Detection was performed using horseradish peroxidase-conjugated Goat Anti-Mouse IgG antibody.		Fig. 3. Staining of formalin-fixed, paraffin-embedded rat jejunum. Stained with Human Anti-BrdU antibody (HCA321) in the nucleus of paraffin-embedded rat jejunum treated with 100 mg/kg BrdU in PBS. Detection was performed using an HRP-conjugated secondary antibody.
Fig. 4. Staining of a cryosection of mouse lymph node. Stained with Rat Anti-Mouse CD11b antibody (MCA74G). Detection was performed by horseradish peroxidase-conjugated Goat Anti-Rat IgG antibody.

Frequently Asked Questions

References

• Engel K et al. (2011). Effects of preanalytical variables on the detection of proteins by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med 135, 537–543
• McLean IW and Nakane PK (1974). Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem 22, 1077–1083
• Pollard K et al. (1987). Fixation, processing, and immunochemical reagent effects on preservation of T-lymphocyte surface membrane antigens in paraffin-embedded tissue. J Histochem Cytochem 35, 1329–1338

Further Reading

• Antigen Retrieval Method for Cryostat Frozen Sections
• Antigen Retrieval Method for Frozen Sections