**6.1 Kind of dilution buffers**

Fifteen monoclonal antibodies were diluted in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl (PBS), 10 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl (TBS), or 10 mM FEPES-NaOH buffer (pH 7.4) containing 150 mM NaCl (HBS); 1% bovine serum albumin (BSA) (final concentration) was added to each solution. Paraffin sections from mouse tissues fixed with formaldehyde were immunostained after autoclaving in 20 mM Tris-HCl buffer (pH 9.0) for 10 min. The sections were treated with the primary antibodies diluted with the solutions overnight at 4°C and successively with Envision HRR (Dakocytomation) for 1 h at room temperature. As shown in **Table 2**, all the antibodies diluted with TBS showed stronger immunoreactions than those diluted with PBS or HBS [45]. Although the reasons are unclear, the binding of phosphate ion (a larger ion) to positively charged regions of epitopes and paratopes may reduce the flexibility of peptide chains.

## **6.2 Ionic strength of dilution solution**

Fifteen monoclonal antibodies were diluted in 1% BSA/10 mM Tris-HCl buffer (pH 7.4) containing 50 mM NaCl, 150 mM NaCl, or 300 mM NaCl. After autoclaving, the paraffin sections were treated with the primary antibodies overnight and then with Envision HRR for 1 h at room temperature. The results are shown in **Table 2**. Most of the antibodies showed strong immunostaining when they were diluted with a buffer containing 50 mM NaCl [45]. However, monoclonal antibodies to proliferating cell nuclear antigen (PCNA) showed the strongest immunostaining when diluted with a buffer containing 300 mM NaCl, and monoclonal antibodies to glial fibrillary


*FFPE sections (6 μm) of mouse tissues were autoclaved in 20 mM Tris-HCl (pH 9.0) for 10 min and then immunostained with monoclonal antibodies. Antibodies were diluted with 150 mM NaCl/10 mM phosphate buffer (pH 7.4) (PBS), 150 mM NaCl/10 mM Tris-HCl (pH 7.4) (TBS), or 150 mM NaCl/10 mM HEPES buffer (pH 7.4) (HBS). The antibodies were also diluted with 10 mM Tris-HCl (pH 7.4) containing 50 mM, 150 mM, or 300 mM NaCl. Immunostaining was scored as followed: 3, strong; 2, moderate; and 1, weak.*

### **Table 2.**

*Effects of diluents for monoclonal antibodies on immunohistochemistry.*

acidic protein (GFAP) and β-actin diluted with 150 mM yielded the strongest immunostaining. Polyclonal antibodies to nuclear transcription factors such as estrogen receptor (ER)α, androgen receptor (AnR), glucocorticoid receptor, and p300 yielded stronger immunostaining when diluted with a buffer containing 150 or 300 mM NaCl than that using a buffer containing 50 mM NaCl (not shown). These results suggest that the net charges of antibodies and antigens influence the contact of these proteins, and the net neighboring charges of antigens also affect the interactions. Nuclear antigens are associated with highly acidic nucleic acid, and β-actin and GFAP bundles are composed of β-actin and GFAP proteins with acidic isoelectric points. Dilution solution with a high ionic strength may reduce the net charges around the antigens and antigen molecules, allowing antibodies to come in contact with their respective antigens.

#### **6.3 Immunoelectron microscopy**

In immunohistochemistry using HRP-labeled antibodies, the staining intensity can be increased using highly sensitive reaction solutions and a longer enzyme reaction time. However, the intensification of immunoreactions is difficult in the post-embedding method and ultracryotomy using colloidal gold-labeled secondary antibody. Instead, the selection of diluents for the antibodies may be more important for obtaining a high labeling density with a low background staining compared

**47**

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

noelectron microscopy.

**7. Concluding remarks**

**Abbreviations**

AnR androgen receptor BSA bovine serum albumin

NRP neuropilin

DAB 3,3′ diaminobenzidine ERα estrogen receptor α

GFAP glial fibrillary acidic protein γ-GTP γ-glutamyl transpeptidase HIAR heat-induced antigen retrieval HRP horseradish peroxidase HSP heat shock protein

with light microscopy, whereas almost no studies have been performed for immu-

method using specimens fixed with the standardized fixative and embedded in LR-White resin [39, 41]. Glutaraldehyde and osmium tetroxide fixed and eponembedded specimens were also used for a few antibodies. After HIAR, ultrathin sections were immunostained using antibodies diluted in the following solutions: PBS, TBS, 10 mM Tris-HCl (pH 7.4) containing 50 mM NaCl, Can Get Signal A (Toyobo Co.), and Can Get Signal B (Toyobo Co.); 1% BSA (final concentration) was then added to the diluents. In general, diluents that produced strong immunoreactions on light microscopy also produced a high labeling density of colloidal gold-labeled antibody. Anti-claudin-5 polyclonal antibody showed the strongest immunoreaction when it was diluted with Can Get Signal A for both LR-White-embedded materials and epon-embedded materials (**Figure 8D**). Can Get Signal A also showed the strongest immunoreactions when used as the diluent for E-cadherin monoclonal antibody. Monoclonal antibodies to β-catenin, β-actin, and clathrin showed a high labeling density when diluted in TBS. Polyclonal antibody to Tom 20 diluted with 50 mM NaCl/10 mM Tris-HCl (**Figure 6B**) or Can Get Signal B showed strong immunostaining. TBS was a better diluent for colloidal gold-labeled secondary antibodies than PBS.

We examined the effect of diluents of several antibodies for the post-embedding

The main mechanisms of HIAR are cleavage of chemical crosslinks formed by formaldehyde and extend of polypeptides chains to expose epitopes. Highly masked epitopes in heat-stable proteins can be exposed with reduction of disulfide bond followed by heating. Heating also cleaves crosslinks formed by double fixation with glutaraldehyde and osmium tetroxide. The principle of HIAR is applicable for immunoelectron microscopy using the post-embedding and pre-embedding methods, in which tissues are fixed with a standardized fixative described in this text. The association of exposed epitopes and antibodies under a suitable solution

are also important for each immunohistochemical reaction.

CAGSM common antigen of secretory granule membrane

PBS phosphate-buffered saline (10 mM phosphate buffer (pH 7.4)

FFPE formalin-fixed and paraffin embedded

containing 150 mM NaCl)

PCNA proliferating cell nuclear antigen

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

*Immunohistochemistry - The Ageless Biotechnology*

acidic protein (GFAP) and β-actin diluted with 150 mM yielded the strongest immunostaining. Polyclonal antibodies to nuclear transcription factors such as estrogen receptor (ER)α, androgen receptor (AnR), glucocorticoid receptor, and p300 yielded stronger immunostaining when diluted with a buffer containing 150 or 300 mM NaCl than that using a buffer containing 50 mM NaCl (not shown). These results suggest that the net charges of antibodies and antigens influence the contact of these proteins, and the net neighboring charges of antigens also affect the interactions. Nuclear antigens are associated with highly acidic nucleic acid, and β-actin and GFAP bundles are composed of β-actin and GFAP proteins with acidic isoelectric points. Dilution solution with a high ionic strength may reduce the net charges around the antigens and antigen molecules, allowing antibodies to come in contact with their respective

*NaCl. Immunostaining was scored as followed: 3, strong; 2, moderate; and 1, weak.*

*Effects of diluents for monoclonal antibodies on immunohistochemistry.*

**Antibodies Clones and subclasses Dilution buffer type mM NaCl/10 mM TB**

PCNA PC10; IgG2a 1 2 2 1 2 2–3 ERα D12; IgG2a 3 2 2 2 2 2 ERα ID5; IgG1 1 1–2 1 2 1-2 1 S-100 M2A10; IgG1 1 2 1–2 3 2 1 Mortalin JG1; IgG3 3 3 3 3 3 3 HSP 70 sc-27; IgG2a 1 2 2 3 2 1–2 α-Synuclein 42; IgG1 2 2 2 2 2 2 GFAP 6F2; IgG1 2 2 1 1 2 2 Desmin D33; IgG1 1 2 1 1 2 2 β-Actin AC-74; IgG2a 2 2 2 1 2 1–2 Clathrin X22; IgG1 1 2 2–3 3 2 1 E-Cadherin 36B5; Ig1 1 2 2 3 2 1 β-Catenin sc-763; IgG1 1 2 1 3 2 1 γ-GTP 5B9; IgG1 1 2 2 3 2 1 CASGM 170-5; IgG1 2 2 2 3 2 1 *FFPE sections (6 μm) of mouse tissues were autoclaved in 20 mM Tris-HCl (pH 9.0) for 10 min and then immunostained with monoclonal antibodies. Antibodies were diluted with 150 mM NaCl/10 mM phosphate buffer (pH 7.4) (PBS), 150 mM NaCl/10 mM Tris-HCl (pH 7.4) (TBS), or 150 mM NaCl/10 mM HEPES buffer (pH 7.4) (HBS). The antibodies were also diluted with 10 mM Tris-HCl (pH 7.4) containing 50 mM, 150 mM, or 300 mM* 

**PBS TBS HBS 50 150 300**

In immunohistochemistry using HRP-labeled antibodies, the staining intensity can be increased using highly sensitive reaction solutions and a longer enzyme reaction time. However, the intensification of immunoreactions is difficult in the post-embedding method and ultracryotomy using colloidal gold-labeled secondary antibody. Instead, the selection of diluents for the antibodies may be more important for obtaining a high labeling density with a low background staining compared

**46**

antigens.

**Table 2.**

**6.3 Immunoelectron microscopy**

with light microscopy, whereas almost no studies have been performed for immunoelectron microscopy.

We examined the effect of diluents of several antibodies for the post-embedding method using specimens fixed with the standardized fixative and embedded in LR-White resin [39, 41]. Glutaraldehyde and osmium tetroxide fixed and eponembedded specimens were also used for a few antibodies. After HIAR, ultrathin sections were immunostained using antibodies diluted in the following solutions: PBS, TBS, 10 mM Tris-HCl (pH 7.4) containing 50 mM NaCl, Can Get Signal A (Toyobo Co.), and Can Get Signal B (Toyobo Co.); 1% BSA (final concentration) was then added to the diluents. In general, diluents that produced strong immunoreactions on light microscopy also produced a high labeling density of colloidal gold-labeled antibody. Anti-claudin-5 polyclonal antibody showed the strongest immunoreaction when it was diluted with Can Get Signal A for both LR-White-embedded materials and epon-embedded materials (**Figure 8D**). Can Get Signal A also showed the strongest immunoreactions when used as the diluent for E-cadherin monoclonal antibody. Monoclonal antibodies to β-catenin, β-actin, and clathrin showed a high labeling density when diluted in TBS. Polyclonal antibody to Tom 20 diluted with 50 mM NaCl/10 mM Tris-HCl (**Figure 6B**) or Can Get Signal B showed strong immunostaining. TBS was a better diluent for colloidal gold-labeled secondary antibodies than PBS.
