**3. Mechanisms of chemical fixation**

Chemical fixatives used for immunohistochemistry are limited to formaldehyde and glutaraldehyde. Formaldehyde is used for tissue fixation in both light and electron microscopy, while glutaraldehyde is used as a fixative only in electron microscopy. Although formaldehyde and glutaraldehyde are popular fixatives for histology and pathology, the characteristics and fixation mechanisms are assumed to be quite different. Since formalin is composed of about 35% formaldehyde aqueous solution containing about 10% methanol to prevent the polymerization of formaldehyde and is usually diluted 10-fold as 10% formalin, its fixation mechanisms should be the same as those for 4% formaldehyde.

### **3.1 Formaldehyde**

The mechanism of fixation using formaldehyde is thought to be as follows. Formaldehyde forms an adduction of hydroxymethyl/methylol (CH2OH) to functional groups of amino acids (such as lysine, arginine, and cysteine) (**Figure 1a, d**, and **f**), the N-terminus of polypeptides, and bases of RNA and single strand DNA [7, 8]. A part of the methylol group of lysine and arginine forms imines (Schiff base) through the removal of H2O (**Figure 1a** and **d**), and the imines of lysine then combine with the side chains of amino acids, such as tyrosine (**Figure 1b**), tryptophan (**Figure 1c**), asparagine, glutamine, and histidine, and the imine of arginine (**Figure 1e**) to form approximately 0.25-nm methylene bridges (-CH2-). Although lysines are reported to be major reactive residues for formaldehyde in native proteins, only lysines located on the surface area are modified by formaldehyde [9]. Methylols of cysteine form methylene

*Reaction of formaldehyde with proteins.*

bridges with tyrosine (**Figure 1g**), arginine, and the N-terminus of peptides. These adductions, imine formations, and crosslinks progress in a time-dependent and temperature-dependent manner. Although formaldehyde is rapidly and freely permeable into cells and tissues blocks, the chemical reactions are relatively slow. Fox et al. reported that the binding of 14C formaldehyde to 16 μm of fresh frozen sections only reached a plateau after 24 h at 25°C [10].

Although formaldehyde forms intra- and intermolecular crosslinks in proteins, the tertiary structures of the proteins are almost completely preserved [9, 11]. The methylene bridges between lysine and the phenyl residue of tyrosine are stable but most methylene bridges are unstable and reversible. Since basic residues of amino acids are modified with formaldehyde and the isoelectric point of proteins shifts to acidic, basic proteins should be precipitated at around the pH of the buffer (pH 7.2– 7.4) used to dissolve the formaldehyde, based on the principle of isoelectric precipitation. Formaldehyde may first produce crosslinks among proteins in relatively stable core complexes, such as cell organelles, filament proteins in the cytoplasm

**33**

**Figure 2.**

*protein by osmium tetroxide post-fixation.*

*Antigen Retrieval for Light and Electron Microscopy DOI: http://dx.doi.org/10.5772/intechopen.80837*

**3.2 Glutaraldehyde**

both heat and acid treatments [16].

and extracellular matrix, and chromatin, and then soluble proteins attach to these complexes to form a gel-like structure. Thus, these crosslinks interfere with the access of antibodies to antigens even if the epitopes do not have functional groups of amino acids that are directly modified by formaldehyde, as demonstrated in the model system by Sompran et al. using peptide epitopes coupled to glass slides [12]. FFPE specimens are assumed to be highly cross-linked, compared with formalinfixed frozen sections. Since ethanol accelerates the imine formation of methylol groups and causes the rearrangement of the β-sheet of polypeptides and the exposure of hydrophobic amino acids, which are hidden in aqueous solutions, both intraand intermolecular crosslinking should advance during dehydration and clearing with ethanol and xylene [13], and immersion in paraffin at around 60°C facilitates further crosslinking. On the other hand, some antigens in cell organelles might come in contact with antibodies more easily than those in frozen sections because membrane lipids are extracted and barriers are destroyed during dehydration.

Glutaraldehyde has been widely used as the standard primary fixative for electron microscopy specimens since introduced by Sabatini et al. in 1963 [14]. A mixture of glutaraldehyde and formaldehyde is also a popular fixative for cytology, enzyme cytochemistry, and immunoelectron microscopy. Glutaraldehyde (**Figure 2a**) has two aldehydes that can directly crosslink with the ε-amino residues of lysine and the N-terminus of polypeptides by forming a Schiff base. However, most investigators think that the rapid and extremely stable crosslinks formed by glutaraldehyde are based on the oligomeric form of glutaraldehyde. Kawahara et al. demonstrated that protein crosslinkage by forming the Schiff base and the aldol condensation of glutaraldehyde monomers occur almost in parallel and result in the formation of a linear glutaraldehyde oligomer with several Schiff base linkages branching off forming (-CH=CH-CH=N-R)n (**Figure 2b**), since glutaraldehyde solution showed no absorbance at 235 nm caused by α,β-unsaturated bonds in the absence of amines [15]. The resulting resonance structures are extremely stable to

*Reaction of glutaraldehyde and osmium tetroxide with proteins and effect of heating. F-protein, fragmented* 

*Antigen Retrieval for Light and Electron Microscopy DOI: http://dx.doi.org/10.5772/intechopen.80837*

and extracellular matrix, and chromatin, and then soluble proteins attach to these complexes to form a gel-like structure. Thus, these crosslinks interfere with the access of antibodies to antigens even if the epitopes do not have functional groups of amino acids that are directly modified by formaldehyde, as demonstrated in the model system by Sompran et al. using peptide epitopes coupled to glass slides [12].

FFPE specimens are assumed to be highly cross-linked, compared with formalinfixed frozen sections. Since ethanol accelerates the imine formation of methylol groups and causes the rearrangement of the β-sheet of polypeptides and the exposure of hydrophobic amino acids, which are hidden in aqueous solutions, both intraand intermolecular crosslinking should advance during dehydration and clearing with ethanol and xylene [13], and immersion in paraffin at around 60°C facilitates further crosslinking. On the other hand, some antigens in cell organelles might come in contact with antibodies more easily than those in frozen sections because membrane lipids are extracted and barriers are destroyed during dehydration.

### **3.2 Glutaraldehyde**

*Immunohistochemistry - The Ageless Biotechnology*

bridges with tyrosine (**Figure 1g**), arginine, and the N-terminus of peptides. These adductions, imine formations, and crosslinks progress in a time-dependent and temperature-dependent manner. Although formaldehyde is rapidly and freely permeable into cells and tissues blocks, the chemical reactions are relatively slow. Fox et al. reported that the binding of 14C formaldehyde to 16 μm of fresh frozen sections only

Although formaldehyde forms intra- and intermolecular crosslinks in proteins, the tertiary structures of the proteins are almost completely preserved [9, 11]. The methylene bridges between lysine and the phenyl residue of tyrosine are stable but most methylene bridges are unstable and reversible. Since basic residues of amino acids are modified with formaldehyde and the isoelectric point of proteins shifts to acidic, basic proteins should be precipitated at around the pH of the buffer (pH 7.2– 7.4) used to dissolve the formaldehyde, based on the principle of isoelectric precipitation. Formaldehyde may first produce crosslinks among proteins in relatively stable core complexes, such as cell organelles, filament proteins in the cytoplasm

reached a plateau after 24 h at 25°C [10].

*Reaction of formaldehyde with proteins.*

**32**

**Figure 1.**

Glutaraldehyde has been widely used as the standard primary fixative for electron microscopy specimens since introduced by Sabatini et al. in 1963 [14]. A mixture of glutaraldehyde and formaldehyde is also a popular fixative for cytology, enzyme cytochemistry, and immunoelectron microscopy. Glutaraldehyde (**Figure 2a**) has two aldehydes that can directly crosslink with the ε-amino residues of lysine and the N-terminus of polypeptides by forming a Schiff base. However, most investigators think that the rapid and extremely stable crosslinks formed by glutaraldehyde are based on the oligomeric form of glutaraldehyde. Kawahara et al. demonstrated that protein crosslinkage by forming the Schiff base and the aldol condensation of glutaraldehyde monomers occur almost in parallel and result in the formation of a linear glutaraldehyde oligomer with several Schiff base linkages branching off forming (-CH=CH-CH=N-R)n (**Figure 2b**), since glutaraldehyde solution showed no absorbance at 235 nm caused by α,β-unsaturated bonds in the absence of amines [15]. The resulting resonance structures are extremely stable to both heat and acid treatments [16].

#### **Figure 2.**

*Reaction of glutaraldehyde and osmium tetroxide with proteins and effect of heating. F-protein, fragmented protein by osmium tetroxide post-fixation.*

Since the cross-linked proteins rapidly form harder gel-like structures, compared with those created by formaldehyde, only thin layers of tissues can be fixed well using immersion fixation. Since aldehyde residues remain in the tissues fixed with glutaraldehyde, the aldehyde should be quenched using amides, such as glycine, ammonium chloride, and tris(hydroxymethyl) aminomethane, or reduced to alcohols using sodium borohydride prior to immunostaining.

#### **3.3 Osmium tetroxide**

Since osmium tetroxide binds to the unsaturated bonds of fatty acids and fixes membrane lipids, providing contrast by scattering electron beams, it is used as the post-fixing reagent after glutaraldehyde fixation in electron microscopy. Osmium tetroxide should also bind to the carbon-carbon double bonds formed by glutaraldehyde fixation (**Figure 2c**). However, since osmium tetroxide cleaves polypeptides in tryptophan residues and oxidizes methionine to methionine sulfone and cysteine to cysteic acid [17], osmium tetroxide significantly inhibits immunoreactions.
