**5.3 Post-embedding method**

The post-embedding method provides more reproducible and reliable immunostaining results than the pre-embedding method, which produces a limited and heterogeneous penetration of antibodies into the tissues, since immunoreactions occur on the surfaces of ultrathin sections. Furthermore, immunoreactions on ultrathin sections permit the counting of immunogold particles, enabling semi-quantitative analyses, and the simultaneous staining of multiple antigens. Although the post-embedding method has these advantages, it has only been used for a limited number of antigens because antigenicity is frequently lost during the dehydration and embedding procedures. Specimens embedded in acrylic resins without osmication show a disrupted membrane structure and poorly contrasted cell organelles.

#### **Figure 4.**

*HIAR in frozen sections form specimens fixed with the standardized fixative. Mouse tissues were fixed with the standardized fixative and immersed in 10, 15, and 20% sucrose and then frozen. Frozen sections (15 μm) were immunostained with anti-E-cadherin monoclonal antibody in the small intestine (A and B), anti-β-catenin monoclonal antibody in the pancreas (C and D), and anti-claudin 5 polyclonal antibody in the kidney (E and F) after heating in 10 mM Tris-HCl buffer (pH 9.0) containing 10% sucrose for 3 h at 70°C (B, D, and F) or without heating (A, C, and E). Endogenous mouse immunoglobulins are seen in the lamina propria mucosa (arrows) and plasma cells (outline arrows), but E-cadherin immunoreactions is not detected without heating (A). However, the endogenous immunoglobulin immunostaining is negative and E-cadherin is clearly localized along the lateral membrane of intestinal epithelium after heating; the junctional complexes show strong immunoreactions (B). β-Catenin immunoreaction is present along the lateral membrane of pancreatic acinar cells (D) but not in the section without heating (C). After heating, claudin-5 is localized in the glomeruli and distal tubules in the kidney (F). Bar = 50 μm.*

Several investigators have applied HIAR to the post-embedding method and have reported its usefulness [37–39]. Tissues were fixed with a mixture of formaldehyde and glutaraldehyde or formaldehyde alone and embedded in acryl resins; then, ultrathin sections were heated in various solutions. We attempted to establish a standardized method for immunoelectron microscopy that would satisfy the following requirements: (1) the preservation of fine cell structures with good image contrast in tissues embedded in acryl resins without OsO4 post-fixation, (2) the application of HIAR to obtain a high labeling density, and (3) a simple and reproducible method that does not require special equipment [32, 39].

Tissues were fixed with the standardized fixative and dehydrated with dimethylformamide (DMF) on ice and embedded in the LR-White resin, since DMF may reduce abrupt osmotic pressure changes in the tissues and the extraction of membrane lipids. The resin was polymerized for 24 h at 55°C. Ultrathin sections mounted on a nickel grid were heated in 0.5 M Tris-HCl buffer (pH 9.0) for 1–2 h at 95°C. After immunogold labeling, the sections were treated with 2% glutaraldehyde containing 0.05% tannic acid in 0.1 M phosphate buffer (pH 5.5) for 5 min and with 1% OsO4/0.1 M phosphate buffer (pH 7.4) for 5 min and then double stained with uranyl acetate and lead citrate. This method yielded strong and reproducible immunoreactions for many soluble, membrane bound, and filamentous proteins (**Figure 6**), [32, 39]. Furthermore, tannic

**41**

**Figure 6.**

**Figure 5.**

acid treatment followed by osmium tetroxide treatment produced good contrasted images. The cellular membranes produced a positive image and the cell organelles, such as mitochondria, the Golgi complexes, secretory granules, and lysosomes, were well

*HIAR for immunoelectron microscopy with the post-embedding method. Mouse kidney was fixed with the standardized fixative and then embedded in LR-White resin. Ultrathin sections were heated in 0.5 M Tris-HCl (pH 9.0) for 1 h at 95°C and treated with anti-β-actin/TBS (A), Tom 20/10 mM Tris-HCl (pH 7.4) containing 50 mM NaCl (B), and anti-mortalin (mitochondrial 70 kD heat shock protein)/TBS (C) and then treated with colloidal gold-labeled secondary antibodies/TBS. A. Strong β-actin immunoreactions are seen in the foot processes of podocyte (P) (outline arrows) and in the cytoplasm of mesangial cell (M). Lymphocyte (L) shows potty reactions beneath the cell membrane (arrows). B. Tom 20 is localized along the mitochondria but not recognized in the lysosome (outline arrow). C. Mitochondria show mortalin immunoreaction, whereas lysosomes (outline arrows), apical canaliculi (arrows), and nucleus are negative for staining. Bars = 500 nm.*

*HIAR for immunoelectron microscopy with the pre-embedding method. Frozen sections immunostained with anti-E-cadherin antibody (A) and anti-claudin-5 antibody (B) demonstrated in* **Figure 4** *were post-fixed with osmium tetroxide, dehydrated in ethanol, and then embedded in the epoxy resin. A. Immunoreaction for E-cadherin is seen in the adherence junction (outline arrows) and spotty reaction (arrows) is present in the lateral membrane of intestinal epithelium. B. Claudin-5 is localized in the foot processes of podocyte (arrows)* 

*in the renal glomerulus: P, podocyte; E, endothelial cells; and M, mesangial cell. Bar = 2 μm.*

*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*

#### **Figure 5.**

*Immunohistochemistry - The Ageless Biotechnology*

Several investigators have applied HIAR to the post-embedding method and have reported its usefulness [37–39]. Tissues were fixed with a mixture of formaldehyde and glutaraldehyde or formaldehyde alone and embedded in acryl resins; then, ultrathin sections were heated in various solutions. We attempted to establish a standardized method for immunoelectron microscopy that would satisfy the following requirements: (1) the preservation of fine cell structures with good image contrast in tissues embedded in acryl resins without OsO4 post-fixation, (2) the application of HIAR to obtain a high labeling density, and (3) a simple and repro-

*HIAR in frozen sections form specimens fixed with the standardized fixative. Mouse tissues were fixed with the standardized fixative and immersed in 10, 15, and 20% sucrose and then frozen. Frozen sections (15 μm) were immunostained with anti-E-cadherin monoclonal antibody in the small intestine (A and B), anti-β-catenin monoclonal antibody in the pancreas (C and D), and anti-claudin 5 polyclonal antibody in the kidney (E and F) after heating in 10 mM Tris-HCl buffer (pH 9.0) containing 10% sucrose for 3 h at 70°C (B, D, and F) or without heating (A, C, and E). Endogenous mouse immunoglobulins are seen in the lamina propria mucosa (arrows) and plasma cells (outline arrows), but E-cadherin immunoreactions is not detected without heating (A). However, the endogenous immunoglobulin immunostaining is negative and E-cadherin is clearly localized along the lateral membrane of intestinal epithelium after heating; the junctional complexes show strong immunoreactions (B). β-Catenin immunoreaction is present along the lateral membrane of pancreatic acinar cells (D) but not in the section without heating (C). After heating, claudin-5 is localized in the glomeruli and* 

Tissues were fixed with the standardized fixative and dehydrated with dimethylformamide (DMF) on ice and embedded in the LR-White resin, since DMF may reduce abrupt osmotic pressure changes in the tissues and the extraction of membrane lipids. The resin was polymerized for 24 h at 55°C. Ultrathin sections mounted on a nickel grid were heated in 0.5 M Tris-HCl buffer (pH 9.0) for 1–2 h at 95°C. After immunogold labeling, the sections were treated with 2% glutaraldehyde containing 0.05% tannic acid in 0.1 M phosphate buffer (pH 5.5) for 5 min and with 1% OsO4/0.1 M phosphate buffer (pH 7.4) for 5 min and then double stained with uranyl acetate and lead citrate. This method yielded strong and reproducible immunoreactions for many soluble, membrane bound, and filamentous proteins (**Figure 6**), [32, 39]. Furthermore, tannic

ducible method that does not require special equipment [32, 39].

**40**

**Figure 4.**

*distal tubules in the kidney (F). Bar = 50 μm.*

*HIAR for immunoelectron microscopy with the pre-embedding method. Frozen sections immunostained with anti-E-cadherin antibody (A) and anti-claudin-5 antibody (B) demonstrated in* **Figure 4** *were post-fixed with osmium tetroxide, dehydrated in ethanol, and then embedded in the epoxy resin. A. Immunoreaction for E-cadherin is seen in the adherence junction (outline arrows) and spotty reaction (arrows) is present in the lateral membrane of intestinal epithelium. B. Claudin-5 is localized in the foot processes of podocyte (arrows) in the renal glomerulus: P, podocyte; E, endothelial cells; and M, mesangial cell. Bar = 2 μm.*

#### **Figure 6.**

*HIAR for immunoelectron microscopy with the post-embedding method. Mouse kidney was fixed with the standardized fixative and then embedded in LR-White resin. Ultrathin sections were heated in 0.5 M Tris-HCl (pH 9.0) for 1 h at 95°C and treated with anti-β-actin/TBS (A), Tom 20/10 mM Tris-HCl (pH 7.4) containing 50 mM NaCl (B), and anti-mortalin (mitochondrial 70 kD heat shock protein)/TBS (C) and then treated with colloidal gold-labeled secondary antibodies/TBS. A. Strong β-actin immunoreactions are seen in the foot processes of podocyte (P) (outline arrows) and in the cytoplasm of mesangial cell (M). Lymphocyte (L) shows potty reactions beneath the cell membrane (arrows). B. Tom 20 is localized along the mitochondria but not recognized in the lysosome (outline arrow). C. Mitochondria show mortalin immunoreaction, whereas lysosomes (outline arrows), apical canaliculi (arrows), and nucleus are negative for staining. Bars = 500 nm.*

acid treatment followed by osmium tetroxide treatment produced good contrasted images. The cellular membranes produced a positive image and the cell organelles, such as mitochondria, the Golgi complexes, secretory granules, and lysosomes, were well

contrasted. Nucleic acids (chromatins, nucleoli, and ribosomes), intracellular filaments (actin filaments, 10 nm filaments, and microtubules), and collagen fibers were well visualized.

### **5.4 Osmicated and epon-embedded specimens**

Archives of materials embedded in epoxy resins are collected in many histology and pathology laboratories and in hospitals for morphological analyses, and these archives are expected to provide valuable data if they can be used for immunohistochemical studies. However, conventionally processed specimens for transmission electron microscopy have been regarded as unsuitable for immunoelectron microscopy using post-embedding methods for the following reasons: (1) Glutaraldehyde significantly suppresses antigen-antibody interactions and HIAR is ineffective for most antigens. (2) Osmium tetroxide severely inhibits immunoreactions by cleaving polypeptides and oxidizing methionine and cysteine [17]. (3) Epoxy resins produce tight three-dimensional crosslinks that suppress antigen-antibody interactions.

A limited number of antigens can be successfully detected on sections from conventionally processed materials. One method involves the oxidation and removal of osmium by treating ultrathin sections with sodium metaperiodate aqueous solution [40]. Another method involves the partial removal of epoxy resins by treating the sections with hydrogen peroxide or sodium/potassium ethoxide [41, 42]. The combined use of partial deresination and heat treatment has also been examined. Borson and Skjorten polymerized the epoxy resin to reduce copolymerization between the proteins and the resin and to make a porous polymer for applying HIAR to glutaraldehyde-fixed and epon-embedded materials [43]. We have also demonstrated that HIAR is effective for post-embedding immunoelectron microscopy using conventionally processed epon blocks, contrary to presumptions that antigen detection would be a special case in these specimens [44].

#### *5.4.1 Frozen sections fixed with glutaraldehyde and osmium tetroxide*

The effectiveness of HIAR in the post-embedding method using conventionally processed epon-embedded specimens was systematically examined for 18 antibodies. Frozen sections fixed with 2% glutaraldehyde for 30 min at room temperature or with 2% glutaraldehyde for 30 min followed by 1% osmium tetroxide for 30 min at room temperature were dehydrated with ethanol and then rehydrated to elucidate the validity of HIAR for avoiding the effects of epoxy resin embedment. In another experiment, frozen sections fixed with glutaraldehyde and osmium tetroxide were treated with sodium metaperiodate, since this reagent has been reported to be effective for osmicated materials, as described above.

After autoclaving in Tris-HCl buffer (pH 9.0), 7 of the 18 antibodies exhibited a strong immunoreaction in frozen sections fixed with glutaraldehyde and osmium tetroxide, whereas heating revealed almost no effect on the sections fixed with glutaraldehyde alone (**Table 1**; **Figure 7**). Treatment with sodium metaperiodate was ineffective for the antigen retrieval of all the antibodies (**Table 1**). The mechanisms of HIAR in the frozen sections fixed with glutaraldehyde and osmium tetroxide were assumed to be as follows [44]. Osmium tetroxide binds to ethylene bonds formed by glutaraldehyde fixation, i.e., -CH=CH-CH=N-R (**Figure 2c**). Heat treatment removes the osmium tetroxide additives and forms 1, 2-diols (**Figure 2d**), since the black color fades in frozen sections fixed with glutaraldehyde and osmium tetroxide after autoclaving. The cleaving of the double bonds should extend the antigen polypeptides and increase the flexibility of the polypeptides. However, the morphology in the frozen tissues fixed with glutaraldehyde and osmium

**43**

**Table 1.**

*tetroxide.*

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

*5.4.2 HIAR in epon-embedded materials*

glutaraldehyde.

tetroxide was more disrupted after autoclaving, compared with those fixed with glutaraldehyde alone or 4% formaldehyde containing 25 mM CaCl2. The reason may be as follows. Osmium tetroxide treatment induces polypeptide fragmentation (**Figure 2c** and **d**), and heating extracts the fragments after crosslink cleavage by

Partial deresination with sodium ethoxide was required for light microscopy using semi-thin epon sections. After autoclaving in 100 m Tris-HCl (pH 9.0) for 10 min, six of the seven antibodies that showed positive immunoreactions in the frozen sections exhibited clear localizations in the semi-thin sections. α-Amylase (**Figure 8A** and **B**), clathrin (**Figure 8C**), and claudin-5 (**Figure 8D**), which all showed positive immunoreactions in the semi-thin sections, were localized on ultrathin sections using colloidal gold-labeled antibodies. For HIAR, ultrathin sections were heated in 500 mM Tris-HCl buffer (pH 9.0) for 1–3 h at 95°C. Although

> **GA-EtOH GA-OsO4-EtOH Non Autoclave Non NaIO4 Autoclave**

heat treatment was essential for the detection of antigens on ultrathin sections, heat treatment reduced the electron density of ribosomes, chromatins,

PCNA 0 1 0 0 2 Clathrin 0 0–1 0 0 3 GFAP 0 0–1 0 0 3 Occludin 0 0–1 0 0 3 Tom 20 0 0–1 0 0 3 Claudin-5 0 0 0 0 2 α-Amylase 0–1 1 0 0–1 2 NRP1 0 0 0 1 β-Catenin 0–1 0 0 0 E-Cadherin 0 0–1 0 0 Desmin 0 0–1 0 0 Caveolin 0 0 0 0 VEGFR2 0–1 0 0 0 ERα 0 1–2 0 0–1 AnR 0 0 0 0 β-Actin 1 1 0 0 γ-GTP 1 1 0 0 *Fresh frozen sections were fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) for 30 min (GA-EtOH) or fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) for 30 min and further fixed with 1% osmium tetroxide/0.1 M phosphate buffer (pH 7.4) for 30 min (GA-OsO4-EtOH). Some sections fixed with glutaraldehyde and osmium tetroxide were further treated with 1% sodium metaperiodate aqueous solution for 5 min (NaIO4): all sections were hydrated with ethanol and then rehydrated after fixation. The fixed sections were immunostained after autoclaving in 20 mM Tris-HCl (pH 9.0) at 120°C for 10 min (autoclave) or without heat treatment (Non).* 

*Immunostaining was scored as followed: 3, strong; 2, moderate; 1, weak; 0–1, faint; and 0, negative.*

*HIAR in frozen sections prepared from specimens fixed with glutaraldehyde or glutaraldehyde and osmium* 

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

*Immunohistochemistry - The Ageless Biotechnology*

**5.4 Osmicated and epon-embedded specimens**

a special case in these specimens [44].

tive for osmicated materials, as described above.

*5.4.1 Frozen sections fixed with glutaraldehyde and osmium tetroxide*

The effectiveness of HIAR in the post-embedding method using conventionally processed epon-embedded specimens was systematically examined for 18 antibodies. Frozen sections fixed with 2% glutaraldehyde for 30 min at room temperature or with 2% glutaraldehyde for 30 min followed by 1% osmium tetroxide for 30 min at room temperature were dehydrated with ethanol and then rehydrated to elucidate the validity of HIAR for avoiding the effects of epoxy resin embedment. In another experiment, frozen sections fixed with glutaraldehyde and osmium tetroxide were treated with sodium metaperiodate, since this reagent has been reported to be effec-

After autoclaving in Tris-HCl buffer (pH 9.0), 7 of the 18 antibodies exhibited a strong immunoreaction in frozen sections fixed with glutaraldehyde and osmium tetroxide, whereas heating revealed almost no effect on the sections fixed with glutaraldehyde alone (**Table 1**; **Figure 7**). Treatment with sodium metaperiodate was ineffective for the antigen retrieval of all the antibodies (**Table 1**). The

mechanisms of HIAR in the frozen sections fixed with glutaraldehyde and osmium tetroxide were assumed to be as follows [44]. Osmium tetroxide binds to ethylene bonds formed by glutaraldehyde fixation, i.e., -CH=CH-CH=N-R (**Figure 2c**). Heat treatment removes the osmium tetroxide additives and forms 1, 2-diols (**Figure 2d**), since the black color fades in frozen sections fixed with glutaraldehyde and osmium tetroxide after autoclaving. The cleaving of the double bonds should extend the antigen polypeptides and increase the flexibility of the polypeptides. However, the morphology in the frozen tissues fixed with glutaraldehyde and osmium

visualized.

contrasted. Nucleic acids (chromatins, nucleoli, and ribosomes), intracellular filaments (actin filaments, 10 nm filaments, and microtubules), and collagen fibers were well

Archives of materials embedded in epoxy resins are collected in many histology and pathology laboratories and in hospitals for morphological analyses, and these archives are expected to provide valuable data if they can be used for immunohistochemical studies. However, conventionally processed specimens for transmission electron microscopy have been regarded as unsuitable for immunoelectron microscopy using post-embedding methods for the following reasons: (1) Glutaraldehyde significantly suppresses antigen-antibody interactions and HIAR is ineffective for most antigens. (2) Osmium tetroxide severely inhibits immunoreactions by cleaving polypeptides and oxidizing methionine and cysteine [17]. (3) Epoxy resins produce tight three-dimensional crosslinks that suppress antigen-antibody interactions. A limited number of antigens can be successfully detected on sections from conventionally processed materials. One method involves the oxidation and removal of osmium by treating ultrathin sections with sodium metaperiodate aqueous solution [40]. Another method involves the partial removal of epoxy resins by treating the sections with hydrogen peroxide or sodium/potassium ethoxide [41, 42]. The combined use of partial deresination and heat treatment has also been examined. Borson and Skjorten polymerized the epoxy resin to reduce copolymerization between the proteins and the resin and to make a porous polymer for applying HIAR to glutaraldehyde-fixed and epon-embedded materials [43]. We have also demonstrated that HIAR is effective for post-embedding immunoelectron microscopy using conventionally processed epon blocks, contrary to presumptions that antigen detection would be

**42**

tetroxide was more disrupted after autoclaving, compared with those fixed with glutaraldehyde alone or 4% formaldehyde containing 25 mM CaCl2. The reason may be as follows. Osmium tetroxide treatment induces polypeptide fragmentation (**Figure 2c** and **d**), and heating extracts the fragments after crosslink cleavage by glutaraldehyde.
