Heavy Metal Enhancement Technique for Diaminobenzidine in Immunohistochemistry Enables Ultrastructural Observation by Low-vacuum Scanning Electron Microscopy

Abstract

Low-vacuum scanning electron microscopy (LV-SEM) is a powerful tool that allows to observe light microscopic specimens with periodic acid-silver methenamine (PAM) staining at a higher magnification, simply by removing the coverslip. However, it is not suitable for observation of immunohistochemistry (IHC) using 3,3′-diaminobenzidine (DAB) due to insufficient backscattered electron image. Traditional heavy metal enhancement techniques for DAB in IHC, (1) osmium tetroxide and iron, (2) cobalt, (3) methenamine silver (Ag), (4) gold chloride (Gold), and (5) both Ag and Gold (Ag + Gold), were examined by LV-SEM. Tissue specimens from Thy1.1 glomerulonephritis rat kidney stained with α-smooth muscle actin and visualized with DAB were enhanced by each of these enhancement methods. We found, in light microscopic and LV-SEM, that the enhancement with Ag, Gold, or Ag + Gold had better intensity and contrast than others. At a higher magnification, Ag + Gold enhancement showed high intensity and low background, although only Ag or Gold enhancement had nonspecific background. Even after observation by LV-SEM, the quality of specimens was maintained after remounting the coverslip. It was also confirmed that Ag + Gold enhancement could be useful for IHC using clinical human renal biopsy. These findings indicate that Ag + Gold provided an adequate enhancement in IHC for both LM and LV SEM observation.

Introduction

Immunohistochemistry (IHC) has been used vastly in establishing pathological diagnosis of many diseases and organs. Therefore, it is an extremely powerful tool not only for diagnosing diseases but also for elucidating their pathogenesis. Although it is possible to identify the localization and distribution of antigens in IHC tissue specimens under light microscopy, further detailed localization of diseases is crucial to explain the pathogenesis of diseases.

Recently, low-vacuum scanning electron microscopy (LV-SEM) has been developed, and the advantage of LV-SEM is not only its high surface sensitivity and visibility, but also the fact that it can be observed using light microscopic specimens if the backscattered electron image is sufficient. Therefore, the specimens with periodic acid-silver methenamine (PAM) staining can be observed simply by removing the coverslip. LV-SEM does not require complicated sample preparation of transmission electron microscopy (TEM), which can reduce the time required. In addition, samples for light microscopy are usually larger than samples for TEM. Therefore, LV-SEM can not only be used in variety of histological aspect, but also provide detailed information that is useful in discovering the pathogenesis of a disease.

Although LV-SEM could potentially be a superior tool, there are challenges to visualize IHC specimens with 3,3′-diaminobenzidine (DAB) in LV-SEM. One of the challenges is insufficient backscattered electron image of DAB. Traditionally, the enhancement of DAB using heavy metal has been performed in IHC for light microscopic observation.^1–3 In fact, several studies for modification of DAB color by metallic ions were performed as a application for double immunostaining. ¹

In our study, we examined whether several traditional heavy metal enhancement techniques for DAB in IHC, (1) osmium and Fe, ⁴ (2) cobalt (Co), ¹ (3) methenamine silver (Ag), ⁵ (4) gold chloride (Gold), ⁶ and (5) Ag and Gold (Ag + Gold) double enhancement, ⁵ are useful for the observation of IHC by light microscopy and LV-SEM.

Materials and Methods

Preparation of Rat Kidney Tissues and IHC

For paraffin-embedded rat kidney tissues, experimental rat Thy-1.1 glomerulonephritis (GN) was induced in Wister rats by the same method as previously reported. ⁷ Briefly, rat was injected via tail vein with anti-Thy1 (OX-7; Cedarlane Laboratories, Toronto, Ontario, Canada) at a dose of 60 µg IgG/100 g body weight in PBS. The rat kidney was obtained 7 days after induction of Thy-1.1 GN and was fixed in 10% buffered formalin, dehydrated, and embedded in paraffin. In IHC for α-smooth muscle actin (α-SMA) staining in paraffin-embedded rat kidney tissues, heat-mediated antigen retrieval was performed in citrate pH 6.0. Then, the tissue underwent 1-hr incubation with anti-SMA antibody (Agilent Dako; Santa Clara, CA), followed by reaction with polymer. Peroxidase activity was visualized using a liquid DAB substrate. This study was conducted in strict accordance with the recommendations of the regulations for the Care and Use of Laboratory Animals of Nippon Medical School. The protocol was approved by the Nippon Medical School Animal Ethics Committee (Permit Number: 37-10).

Preparation of Clinical Human Kidney Tissues and IHC

For IHC of clinical renal biopsy samples, paraffin-embedded human kidney biopsy samples of IgA nephropathy and membranous nephropathy cases were obtained from Nippon Medical School Hospital. The renal biopsy tissues were fixed in 10% buffered formalin, dehydrated, and were embedded in paraffin. For IgA or IgG immunostaining using paraffin-embedded human kidney tissues, after heat-mediated antigen retrieval was performed in citrate pH 6.0, anti-IgA or anti-IgG antibody (Agilent Dako) was incubated for 1 hr, followed by reaction with polymer. Peroxidase activity was visualized using a liquid DAB substrate. This research was approved by the ethics committee at Nippon Medical School (No. B-2020-120).

Enhancement Methods

After visualization of antigens with DAB, specimens were treated with each of the following enhancement methods for DAB:

1. Osmium tetroxide (OsO₄) and potassium ferrocyanide (Fe) enhancement: Tissues were incubated in 2% OsO₄ for 30 min at room temperature. After washing with running water, followed by deionized water, specimens were incubated in 0.4% C₆FeK₄N₆ and 0.8% HCl for 10 min at room temperature.

2. Co enhancement: During DAB staining, 0.5% CoCl₂ in 0.3% hydrogen peroxide (H₂O₂) was added to the DAB solution at a concentration of 2%. Thereafter, the specimens were treated as same as DAB staining.

3. Ag enhancement: Specimens were incubated for 20 min at 60°C in Ag solution containing 25 ml of 3% methenamine, 2.5 ml of 5% silver nitrate, 22.5 ml of distilled water, and 2.5 ml of 5% sodium tetraborate.

4. Gold enhancement: Specimens were incubated in 0.25% Gold for 5 min at room temperature.

5. Double enhancement with Ag and Gold (Ag + Gold): For enhancement with Ag + Gold, Ag enhancement was first performed following step 3. Then specimens were rinsed with deionized water, placed in deionized water, and Gold enhancement was performed following step 4.

After all specimens were treated with enhancement methods, they underwent nuclear staining with hematoxylin. The specimens were mounted with coverslip using the mounting medium Malinol (Muto Pure Chemical Co., Ltd; Tokyo, Japan) in a usual manner for light microscopic specimens.

Observation by Light Microscopy and LV-SEM

The specimens with PAM staining or IHC with or without each of the several enhancement methods were observed by light microscopy. All findings were compared, including each enhancement effect for DAB in IHC. After observation by light microscopy, all specimens were observed by LV-SEM (Hitachi Tabletop Microscope TM3030PLUS; Hitachi High-Technologies Corp., Tokyo, Japan) after removal of coverslip by incubation in xylene overnight. Following LV-SEM observation, the specimens were remounted with coverslip and the quality of IHC by light microscopy was reevaluated.

Scanning Electron Microscopy/Energy-dispersive X-ray Spectrometry (SEM/EDX) Analysis

EDX is a spectral technique that enables identification and visualization of multiple elements simultaneously. To analyze the elemental components of paraffin sections, IHC specimens with each enhancement using heavy metal without hematoxylin staining were provided. A built-in EDX detector (manufactured by Hitachi High-Technologies Corporation) was used with LV-SEM (TM4000Plus; Hitachi High-Technologies Corp). Elemental analysis was performed using a 15-kV electron beam.

Result

Observation by Light Microscopy and LV-SEM With ×200 Magnification

The serial sections of Thy 1.1 GN kidney specimens were immunostained with α-SMA, which is a marker of smooth muscle cells and known to be expressed in the media of arteries, activated mesangial cells in glomeruli, and pericytes of capillaries and myofibroblasts in interstitium in Thy1.1 GN model. ⁸ The α-SMA visualized with DAB was subsequently enhanced by each enhancement method and examined by light microscopy (Fig. 1). In the unenhanced sections with α-SMA staining using DAB (Fig. 1A), the brown color of DAB was seen in the media of small arteries, in some interstitial cells, and in glomerular mesangial cells. The brown positive staining was mildly enhanced and darkened by OsO₄ + Fe enhancement (Fig. 1C). During Co enhancement, the brown color of DAB changed to dark blue and mildly increased the intensity (Fig. 1D). The enhancements by Gold, Ag, and Ag + Gold had higher intensity than OsO₄ + Fe or Co (Fig. 1B, E, and F). These colors were darker and closer to black. However, Gold enhancement had nonspecific responses to tubular cells and tubular lumen, seen in blue color (Fig. 1E). This might be due to the urinary protein reabsorbed by tubules or cast in tubular lumen. The serial specimens with PAM staining showed intact small arteries and the glomerulus with findings of Thy1.1 GN (Fig. 1H).

Figure 1.

The comparison of light microscopic findings of IHC with or without heavy metal enhancement methods. The light microscopic findings were obtained from IHC of α-SMA in paraffin-embedded specimens of Thy-1.1 glomerulonephritis rat kidney with or without enhancement for DAB. (A) DAB without any enhancement and (B) enhancement with Ag + Gold, (C) with OsO4 + Fe, (D) with Cobalt, (E) with Gold, and (F) with Ag. In IHC with each of the several enhancement methods, although α-SMA-positive small vessels and glomerular mesangial area were clearly enhanced and the intensity increased in all enhancement methods, these enhancement effects were clearly strong especially in Ag + Gold (B), Gold (E), and Ag (F) enhancement. (G) In light microscopy, the Ag + Gold specimens that were removed from coverslip for LV-SEM were remounted on the coverslip and showed the same quality of IHC. (H) The serial specimens with PAM staining could indicate the correlation of light microscopic findings between PAM staining and IHC. Scale bar: 50 µm. Abbreviations: IHC, immunohistochemistry; α-SMA, α-smooth muscle actin; DAB, 3,3′-diaminobenzidine; LV-SEM, low-vacuum scanning electron microscopy; PAM, periodic acid-silver methenamine.

After removing the coverslip of the specimens observed in light microscopy, they were observed in LV-SEM (Fig. 2). The same glomeruli as in Fig. 1 were observed at the same magnification (×200). In the specimens with α-SMA immunostaining without enhancement methods, whitish positive signals could not be observed (Fig. 2A). The enhancement by OsO₄ + Fe and Co did not show apparent positive signals (Fig. 2C and D). In contrast, Gold, Ag, and Ag + Gold enhancement had obvious strong positive signals, represented by whitish signals, in glomeruli and small arteries (Fig. 2B, E, and F).

Figure 2.

The comparison of LV-SEM findings of enhancement methods in IHC. After removal of the coverslip, the same region of Fig. 1 was observed by LV-SEM. (A) α-SMA immunostaining tissue visualized with DAB without any enhancement and (B) enhancement with Ag + Gold, (C) OsO4 + Fe, (D) Cobalt, (E) Gold, and (F) Ag. In IHC with each of the several enhancement methods, α-SMA-positive small vessels and glomerular mesangial cells shown by white color were clearly seen in Ag + Gold (B), Gold (E), and Ag (F) enhancement. Scale bar: 50 µm. Abbreviations: LV-SEM, low-vacuum scanning electron microscopy; IHC, immunohistochemistry; α-SMA, α-smooth muscle actin; DAB, 3,3′-diaminobenzidine.

The damage to the tissue specimens caused by the removal of the coverslip and observation by LV-SEM was investigated. After observation in LV-SEM, the Ag + Gold section was reobserved by light microscopy after remounting the coverslip (Fig. 1G). There were no significant changes observed compared with before the coverslip was removed. It supports that the process of removing and remounting the coverslip and observation by LV-SEM did not cause any damage to the tissue specimens with IHC.

Observation by Light Microscopy and LV-SEM With High Magnification

The glomeruli in the specimens with IHC with Gold, Ag, or Ag + Gold enhancement methods were observed by light microscopy in ×400 magnification (Fig. 3A–C). The positive signals of α-SMA in activated and proliferating mesangial cells were clearly observed in these enhanced specimens although Gold enhancement had nonspecific background signals in and around the mesangiolytic ballooning lesion (Fig. 3B). These three Ag + Gold, Gold, and Ag enhancement specimens were evaluated in detail at higher magnification, ×2000 (Fig. 3D–F) and ×5000 (Fig. 3G–I). In the Ag + Gold, Gold, and Ag enhancement methods in IHC, α-SMA-positive whitish mesangial cells were observed in ×2000 magnification. However, the specimens with each Gold or Ag enhancement had several fine granular nonspecific background particles mainly around the red blood cells (Fig. 3H and I). The Ag + Gold enhancement clearly showed sharp and smooth structure without nonspecific background microparticles (Fig. 3D and G). These findings suggested that Ag + Gold enhancement is the recommended enhancement method for DAB under LV-SEM observation.

Figure 3.

The high magnification findings of LV-SEM from light microscopic specimens with IHC. By light microscopy, α-SMA-positive activated and proliferative mesangial cells in glomeruli with Thy 1.1 glomerulonephritis were clearly noted in Ag + Gold (A), Gold (B), and Ag (C) enhancement. Scale bar in A–C: 25 µm. The high magnification findings (×2000: D–F, ×5000: G–I) of the arrowed area of Fig. 3A to C were observed by LV-SEM. In ×2000 magnification, α-SMA-positive mesangial cells shown by white color were clearly seen in Ag + Gold (D), Gold (E), and Ag (F) enhancement. In ×5000 magnification, white color signals were sharper in Ag + Gold (G) than in enhancement of only Gold (H) or Ag (I). The structure of positive signals in Ag + Gold enhancement looked like an actin filament (G). Nonspecific depositions were almost never observed (G). The LV-SEM findings of each of Gold (H) or Ag (I) enhancement showed some nonspecific background, small particles, on mesangial cells and red blood cells. Scale bar in D–F: 20 µm. Scale bar in G–I: 10 µm. Abbreviations: LV-SEM, low-vacuum scanning electron microscopy; IHC, immunohistochemistry; α-SMA, α-smooth muscle actin.

SEM/EDX Analysis

Elemental mapping by SEM/EDX was performed to further investigate and visualize the elemental changes occurring during the enhancement process of Gold after Ag. The IHC specimens with Ag, Gold, and Ag + Gold enhancement were analyzed to visualize Ag and Gold (Fig. 4). Although Ag enhancement had high positive signal of Ag in mesangial area, the background revealed a weak positive signal (Fig. 4C). In gold enhancement, Au (Gold) was mildly positive in the mesangial area with weakly positive in the background (Fig. 4E). Compared with these enhancements, Ag + Gold had high signals of both Ag and Gold, and the background positive signals were relatively lower in both Ag and Au images (Fig. 4A and D). The Ag image of Ag + Gold enhancement showed lower positive signal in the mesangial area than Ag enhancement alone (Fig. 4A), and Au image of Ag + Gold enhancement showed stronger positive signals in mesangial area than Gold enhancement alone (Fig. 4D). It seems to be a well-known effect of “gold tuning.”

Figure 4.

SEM/EDX analysis. Elemental mapping by SEM/EDX was performed to further investigate and visualize the changes of Ag (A–C) and Au (Gold, D–F) in specimens with the Ag + Gold (A and D), Gold (B and E), and Ag (C and F) enhancement, respectively. Ag enhancement had high positive signal of Ag in mesangial area (C), and the background had also some weak positive signals. In gold enhancement, Au (Gold) was mildly positive in the mesangial area and also was weakly positive in the background (E). Compared with these enhancements, Ag + Gold enhancement had high positive signals of both Ag and Gold (A and D), and the background positive signals were relatively low in both Ag and Au images. The Ag image of Ag + Gold enhancement showed relatively lower positive signals in the mesangial area than Ag enhancement alone. In addition, the Au image of Ag + Gold enhancement showed relatively higher positive signals in the mesangial area than Ag enhancement alone. Scale bar: 50 µm. Abbreviation: SEM/EDX, scanning electron microscopy/energy-dispersive X-ray spectrometry.

Observation by Light Microscopy and LV-SEM With Low Magnification

Next, the observations at low magnification in light microscopy and LV-SEM were performed to confirm that LV-SEM can observe wide areas of specimens—one of its most important advantages over TEM. The low magnification areas (×40) were observed using PAM staining or Ag + Gold–enhanced α-SMA immunostaining (Fig. 5). The entirety structure could be observed at low magnification in PAM staining using LV-SEM (Fig. 5B). Using LV-SEM, it was also confirmed that α-SMA immunostaining with Ag + Gold enhancement was also clearly observed in a wide field of view (Fig. 5D) similar to light microscopy (Fig. 5C). These findings confirmed that Ag + Gold enhancement could be useful not only for high magnification observation but also for low magnification in LV-SEM.

Figure 5.

The observation of a wide view of specimens with PAM staining (A and B) and IHC for α-SMA (C and D) by light microscopy (A and C) and LV-SEM (B and D) (×40). After the coverslip was removed, LV-SEM could observe wide area, such as whole histological slide area. Light microscopic and ultrastructural findings of PAM staining and IHC could be compared by light microscopy and LV-SEM. The interest regions in these wide images of the specimens with PAM staining and IHC can be observed by LV-SEM. Scale bar: 150 µm. Abbreviations: PAM, periodic acid-silver methenamine; IHC, immunohistochemistry; α-SMA, α-smooth muscle actin; LV-SEM, low-vacuum scanning electron microscopy.

Clinical Human Kidney Biopsy Samples From IgA Nephropathy and Membranous Nephropathy Cases

The Ag + Gold enhancement method was applied to IHC in clinical diagnosis. Tissue section from IgA nephropathy was stained with anti-IgA antibody–conjugated horseradish peroxidase (HRP), enhanced by Ag + Gold method (Fig. 6A–C). The mesangial IgA deposition was evidently shown in high magnification (×8000) using LV-SEM (Fig. 6C). The specimen from membranous nephropathy was stained with anti-IgG antibody–conjugated HRP, enhanced by Ag + Gold method, and observed in light microscopy and LV-SEM (Fig. 6D–F). The IgG was confirmed to be deposited at the subepithelial region only in high magnification using LV-SEM (Fig. 6F), although light microscopy and low magnification using LV-SEM could only indicate IgG was deposited along with the glomerular basement membrane (GBM) (Fig. 6D).

Figure 6.

Immunohistochemistry for IgA in IgA nephropathy and for IgG in membranous nephropathy in clinical human kidney biopsy specimens. In clinical kidney biopsy, IgA deposition in IgA nephropathy or IgG deposition in membranous nephropathy was observed by light microscopy and LV-SEM. (A–C) Formalin-fixed paraffin-embedded kidney biopsy tissue specimen from IgA nephropathy was stained with anti-IgA antibody, visualized with DAB, and enhanced with Ag + Gold. (A) In light microscopy, IgA-positive signals were seen in mesangial areas. In LV-SEM observation (B: ×2000, C: ×8000), white color IgA-positive signals were clearly detected as paramesangial deposit in mesangial area, as hemispherical deposition. (D–F) Formalin-fixed paraffin-embedded kidney biopsy tissue section from membranous nephropathy was stained with anti-IgG antibody, visualized with DAB, and enhanced with Ag + Gold. (D) In light microscopy, IgG-positive signals were seen diffusely on GBM. In LV-SEM observation (E: ×2000, F: ×8000), white color IgG-positive signals were clearly detected on GBM, especially subepithelial granular IgG deposition on GBM, as membranous nephropathy pattern. Scale bar in A, B, D, and E: 10 µm. Scale bar in C and F: 2.5 µm. Abbreviations: LV-SEM, low-vacuum scanning electron microscopy; DAB, 3,3’-Diaminobenzidine; GBM, glomerular basement membrane.

Discussion

The conventional IHC using DAB could not be observed by LV-SEM due to insufficient backscattered electron image of DAB without heavy metal enhancement for DAB. In the present study, we demonstrated that IHC using DAB with Ag + Gold enhancement could be observed not only by light microscopy but also by LV-SEM at low magnification to high magnification. When IHC is needed for clinical kidney biopsy samples, the Ag + Gold enhancement technique for DAB should be performed in IHC, as sharp and contrast positive signals will be obtained in light microscopy, and ultrastructural fine structures in IHC can be observed by LV-SEM just after removal of the coverslip from light microscopic IHC specimens.

Heavy metals such as OsO₄, Ag, and Gold are known to specifically bind to DAB substrate. ⁹ Using this character, these heavy metals have been used for the enhancement of DAB in IHC. The heavy metals had compositional contrast of backscattered electron that can provide sufficient backscattered electron image for DAB. It is thought that the binding of heavy metal to DAB can provide sufficient backscattered electron image for DAB and enable it to be observed in LV-SEM. Among several enhancement methods using heavy metal, Ag + Gold enhancement showed a high and sharp intensity with low background, which allowed us to identify the exact localization and shape of antigens in IHC in more detail. Furthermore, Ag + Gold enhancement was shown to apply to clinical human kidney biopsy specimens. The deposition of enhanced immunoglobulins was clearly observed in glomeruli in IgA nephropathy and membranous nephropathy under LV-SEM. The enhancement with Ag + Gold was previously reported to be useful for not only IHC in light microscopy but also immuno-TEM observation. ² In the present study, this enhancement technique of Ag + Gold is also very useful for the observation by LV-SEM.

Gold incubation is widely used after the treatment of silver enhancement to prevent occasional loss of the signal by stabilizing or substituting silver with gold.^10,11 Our SEM/EDX data indicated that Gold toned down the Ag signals. There are two possible reasons for it: Gold might replace Ag or gold might cover the Ag and reduce the signal of Ag. In the present study, Gold treatment after Ag enhancement diminished the fine particles in nonspecific background in LV-SEM findings.

The TEM is the strongest tool to observe specimens with high magnification. In a clinical setting, a small part of kidney biopsy sample is separately provided for TEM, and the sample is normally so limited. It is also well known that there are many processes in which time is needed before observation. In addition, strong fixation with changes of antigenicity structure should be performed in the preparation of TEM samples. Therefore, IHC using TEM samples is not generally performed in the diagnosis of clinical renal biopsy. To resolve these problems, LV-SEM is believed to be the comfortable method. LV-SEM had several important advantages, such as simple sample preparation, wide region of observation, findings of high magnification and resolution, and three-dimensional (3D) morphological ultrastructure. LV-SEM can evaluate 3D ultrastructure in the GBM and/or foot process of podocytes in clinical microscopic samples with PAM and/or platinum blue staining. The characteristic LV-SEM findings were reported in IgA nephropathy, Alport syndrome, thin basement membrane disease, minimal change nephrotic syndrome, focal segmental glomerulosclerosis, and membranous nephropathy.^12–16 In addition, we are suggesting a more convenient method for IHC using DAB with Ag + Gold enhancement. By using this enhancement method in IHC, we hope that more precious findings will be obtained in the future.

In conclusion, IHC specimens with Ag + Gold enhancement can be evaluated at high magnification by LV-SEM after simply removing the coverslip from light microscopic specimens. This allows us to compare the findings of light microscopy with that of LV-SEM at higher magnification. Furthermore, using serial sections with PAM staining and IHC enables the comparable observation of both ultrastructural and IHC information in wide areas such as whole slide and at high magnification. These advantages strongly indicate that the Ag and Gold enhancement method in IHC using paraffin-embedded tissue samples is a superior method, as it can be observed at high magnification by LV-SEM.