**4.6 HIAR in frozen sections**

Fresh frozen sections are widely used for immunohistochemical studies, because (1) they preserve antigenicity well, (2) they provide reproducible results because the fixation time is precisely controlled and the fixation is uniform throughout the sections, and (3) they allow antigen localization within a short time for pathological diagnosis. We introduced new fixative, 10% formalin containing 25 mM CaCl2 in 0.1 M HEPES-NaOH buffer (pH 7.4) that is more appropriate for the fixation of fresh frozen sections compared with buffered 10% formalin, because it has a stronger fixation ability and can crosslink proteins more rapidly and stabilize membranes, the extraction of antigens during fixation is minimized, and an excellent tissue structure is maintained after HIAR [29]. After heating in 20 mM Tris-HCl (pH 9.0) for 30 min or in 20 mM citrate buffer (pH 3.0), antigens in the nuclei, other cell organelles, cytoplasm, membranes, and extracellular matrix were clearly visible, even if they showed no immunoreactions without heating.

**37**

**Figure 3.**

**4.7 Antigen retrieval for highly masked epitopes with disulfide bonds**

*DTT (I) or without reduction (H) followed by heating. Bar = 50 μm.*

Few antigens that reduced disulfide bonds react with antibodies when they are analyzed using Western blotting (**Figure 3A**), whereas they reveal negative immunostaining even if the tissues were fixed with formaldehyde or other physical

*Antigen retrieval for highly masked epitopes with disulfide bonds in paraffin and frozen sections. A. VEGFR1 and Tie 2 expressions in the uteri of 10-day-old mice were demonstrated using Western blotting. VEGFR1 and Tie 2 proteins were analyzed after treatment with 2-mercaptoethanol (lines 2 and 4) or without the treatment (lines 1 and 3), respectively. Membrane bound VEGFR1 (150 kD) shows no reaction to the antibody without reduction of disulfide bonds, whereas soluble VEGFR1 (100 kD) binds to the antibody independence of reduction (lines 1 and 2). Tie 2 antibody shows strong immunoreaction to Tie 2 protein-reduced disulfide bonds (lines 3 and 4). B–E. Immunostaining of VEGFR1 (B and C) and Tie 2 (D and E) in the frozen sections of 10-day-old mouse uteri. Fresh frozen sections were fixed with 10% formalin containing 25 mM CaCl2 in 0.1 M HEPES buffer (pH 7.4) for 5 min, treated with 0.1 M dithiothreitol (DTT)/20 mM Tris-HCl (pH 9.0) for 1 h and then with 0.1 M iodoacetamide/0.1 M Tris-HCl (pH 9.0) for 15 min (C and E); B and D, sections without reduction. The sections were then heated in 20 mM Tris-HCl (pH 9.0) for 30 min at 95°C and immunostained with the antibodies. The stroma shows diffuse VEGFR1 immunostaining which may be based on the soluble VEGFR1 in non-reduced section (B), while vasculatures (arrows) and basal membrane of epithelial cells exhibit positive VEGFR1-immunostaining which may be corresponded to the membrane-bound VEGFR1 (C). Clear Tie 2-immunostaining is recognized in the endothelial cells of vasculatures (arrows) in the reduced section (E) but not in the non-reduced section (D). F–I. Immunohistochemistry in paraffin sections. Clathrin was detected in the mouse pancreas after reduction with DTT (G) or without reduction (F) followed by heating in 20 mM Tris-HCl (pH 9.0). Tom 20 was immunostained in the mouse ovary after reduction with* 

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

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

*Immunohistochemistry - The Ageless Biotechnology*

and hydrophilic regions and epitopes. The polypeptide chains then rapidly refold during the cooling process. In tissues, many kinds of proteins with different isoelectric points and molecular weights are tightly packed, and neighboring polypeptides can come in contact with each other. Therefore, epitopes should be concealed during the refolding of the proteins at around a neutral pH because a strong hydrophobic attractive force would randomly entangle the neighboring polypeptides: an electrostatic force may act locally as either an attractive or a repulsive force. At basic or acidic pH values, however, the majority of the extended polypeptides would be charged negatively or positively, and the electrostatic repulsive force would act to prevent random aggregation and entanglement with neighboring polypeptides caused by the hydrophobic force, thereby maintaining a suitable extend conformation for antigen-antibody interactions. When salt is added to the retrieval solutions, the electrostatic force between neighboring polypeptides is canceled, and the hydrophobic attractive force may cause the antigen proteins and neighboring proteins to aggregate, masking the epitopes. Namimatsu et al. reported that heating in citraconic anhydride solution at a neutral pH was useful as a universal antigen retrieval method [27]. Since citraconic anhydride binds to the ε-amino groups of lysine residues re-exposed after heating and places numerous negative charges on proteins, an electric repulsive force may help to keep polypeptides in an unfolded

On the other hand, strong heating at around the isoelectric points of proteins induces their coagulation [2], and increasing the ionic strength also promotes isoelectric precipitation. Therefore, many proteins with neutral isoelectric points can be precipitated in a solution with a neutral pH. The finding that an acidic buffer is effective for some basic antigens probably indicates that these antigens are precipitated by heating in basic buffers [23, 28]. Since heating may destroy the protein conformation, most conformational epitopes associated with noncovalent forces should lose their reactivity to antibodies. On the other hand, HIAR may be effective for conformational epitopes that have been stabilized by

Basic or acidic solutions are effective for HIAR as described above, whereas citrate buffer (pH 6.0) is frequently used in pathological studies. Citrate buffer may be suitable for examining detailed nuclear structures, since heating in basic solutions cleaves and extracts RNAs and reduces the nuclear stainability with hematoxylin. In practice, at least three antigen retrieval solutions at pH 3.0, pH 6.0, and pH 9.0 should be examined when studying the localization of unknown antigens for

Fresh frozen sections are widely used for immunohistochemical studies, because (1) they preserve antigenicity well, (2) they provide reproducible results because the fixation time is precisely controlled and the fixation is uniform throughout the sections, and (3) they allow antigen localization within a short time for pathological diagnosis. We introduced new fixative, 10% formalin containing 25 mM CaCl2 in 0.1 M HEPES-NaOH buffer (pH 7.4) that is more appropriate for the fixation of fresh frozen sections compared with buffered 10% formalin, because it has a stronger fixation ability and can crosslink proteins more rapidly and stabilize membranes, the extraction of antigens during fixation is minimized, and an excellent tissue structure is maintained after HIAR [29]. After heating in 20 mM Tris-HCl (pH 9.0) for 30 min or in 20 mM citrate buffer (pH 3.0), antigens in the nuclei, other cell organelles, cytoplasm, membranes, and extracellular matrix were clearly

visible, even if they showed no immunoreactions without heating.

**36**

state.

disulfide bonds.

the first time.

**4.6 HIAR in frozen sections**

#### **Figure 3.**

*Antigen retrieval for highly masked epitopes with disulfide bonds in paraffin and frozen sections. A. VEGFR1 and Tie 2 expressions in the uteri of 10-day-old mice were demonstrated using Western blotting. VEGFR1 and Tie 2 proteins were analyzed after treatment with 2-mercaptoethanol (lines 2 and 4) or without the treatment (lines 1 and 3), respectively. Membrane bound VEGFR1 (150 kD) shows no reaction to the antibody without reduction of disulfide bonds, whereas soluble VEGFR1 (100 kD) binds to the antibody independence of reduction (lines 1 and 2). Tie 2 antibody shows strong immunoreaction to Tie 2 protein-reduced disulfide bonds (lines 3 and 4). B–E. Immunostaining of VEGFR1 (B and C) and Tie 2 (D and E) in the frozen sections of 10-day-old mouse uteri. Fresh frozen sections were fixed with 10% formalin containing 25 mM CaCl2 in 0.1 M HEPES buffer (pH 7.4) for 5 min, treated with 0.1 M dithiothreitol (DTT)/20 mM Tris-HCl (pH 9.0) for 1 h and then with 0.1 M iodoacetamide/0.1 M Tris-HCl (pH 9.0) for 15 min (C and E); B and D, sections without reduction. The sections were then heated in 20 mM Tris-HCl (pH 9.0) for 30 min at 95°C and immunostained with the antibodies. The stroma shows diffuse VEGFR1 immunostaining which may be based on the soluble VEGFR1 in non-reduced section (B), while vasculatures (arrows) and basal membrane of epithelial cells exhibit positive VEGFR1-immunostaining which may be corresponded to the membrane-bound VEGFR1 (C). Clear Tie 2-immunostaining is recognized in the endothelial cells of vasculatures (arrows) in the reduced section (E) but not in the non-reduced section (D). F–I. Immunohistochemistry in paraffin sections. Clathrin was detected in the mouse pancreas after reduction with DTT (G) or without reduction (F) followed by heating in 20 mM Tris-HCl (pH 9.0). Tom 20 was immunostained in the mouse ovary after reduction with DTT (I) or without reduction (H) followed by heating. Bar = 50 μm.*

### **4.7 Antigen retrieval for highly masked epitopes with disulfide bonds**

Few antigens that reduced disulfide bonds react with antibodies when they are analyzed using Western blotting (**Figure 3A**), whereas they reveal negative immunostaining even if the tissues were fixed with formaldehyde or other physical fixatives followed by heating (**Figure 3B** and **D**). In such cases, the epitopes should be located in a highly folded portion of the antigen protein that may be stabilized with disulfide bonds to form stable secondary and tertiary structures. The reduction of disulfide bonds using dithiothreitol or 2-mercaptoethanol prior to heat treatment yields strong immunoreactions (**Figure 3C** and **E**) [28, 30]. In addition, when epitopes are covered by neighboring heat-resistant polypeptides, the accessibility of antibodies to the epitopes is also inhibited. When immunostaining is performed using old FFEP or using sections on a slide glass stored for a long time, the samples should be oxidized to produce many disulfide bonds in the tissues. The reduction and cleavage of disulfide bonds may be effective for such specimens (**Figure 3F–I**).
