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Abstract Quantitative evaluation of immunohistochemical staining has become a focus of attention in research applications and in pathological diagnosis, such as Her-2/neu assessment in mammary carcinoma. Reproducibility of immunostaining techniques and microscopical evaluation are prerequisites for a standardized and reliable quantitation of immunostaining intensity. In the present study, different staining and microscopical techniques, including fluorescence microscopy, epipolarization microscopy of immunogold-silver, and absorbance microdensitometry were compared concerning suitability for quantitative evaluation. We describe a staining procedure using alkaline phosphatase-based immunohistochemistry with the substrate Vector Red and subsequent microdensitometry with a custom-designed absorbance filter. We have characterized linearity of the staining intensity in dependence of development time, antibody concentration, and section thickness by means of artificial standards consisting of agarose blocks into which immunogold- or alkaline phosphatase-conjugated antibodies were incorporated. Applicability of the different techniques was tested by anti-CD45 immunostaining of leukocytes within rat lung tissue detected by immunofluorescence, immunogold-silver epipolarization microscopy, as well as alkaline phosphatase-based Vector Red absorbance or fluorescence measurement. Excellent qualities of Vector Red for quantitative microdensitometric evaluation of staining intensity were particularly obvious for absorbance microscopy. Applicability in paraffin-embedded tissue as well as in cryosections, excellent segmentation, linearity over a wide range, light stability, and feasibility for permanent mounting and for long-term storage are the outstanding features of this technique for use in routine quantitative evaluation. | |||||||||||||||||
Cellular localization of target substances by immunohistochemistry is a widely used technique and an important tool in research as well as in histopathological diagnosis. 1-3 Qualitative evaluation is, for example, primarily used for the investigation of regulatory processes. 4-6 During the last few years, quantitative evaluation of staining intensity has attracted growing interest in research and diagnosis in the field of pathology. Microdensitometric quantitation has been used in studies investigating breast cancer for proliferation signals and steroid receptor expression, 7-9 as well as in prostate cancer specimens for expression of prostate-specific acid phosphatase. 10 In recent studies, Her-2/neu, a proto-oncogene, which encodes a growth factor receptor of the tyrosine kinase family, has been shown to be overexpressed in 25 to 30% of breast and ovarian malignancies. 11,12 Quantification of Her-2/neu expression or its encoding protein has become an important diagnostic tool for prognosis assessment and prediction of response to targeted gene therapy. 12,13 In situ hybridization and immunohistochemistry have both been shown to be powerful techniques for the evaluation of Her-2/neu expression. 11,12 Numerous techniques used for quantification of mRNAs or proteins are used for investigation of tissue homogenates, thus delivering an averaged result without relation to specific cell types and cellular environment. Immunohistochemistry or in situ hybridization in combination with digital image analysis provide both in situ-localization and objective observer-independent quantitative evaluation. 4 Several techniques of staining and detection for quantitative immunohistochemistry have been described that use enzyme-dependent color reactions as well as fluorescence or epipolarization methods. 5,14-16 Quantification of immunogold-stained targets was predominantly used in electron-microscopic studies. 17-19 Only a few reports have hitherto addressed the quantification of immunogold-silver-enhanced staining intensity by epipolarization imaging in light microscopy. 20,21 This group recently described quantitative evaluation of Cox-1 and Cox-2 immunostaining by image analysis of immunogold-silver-enhanced staining intensity by epipolarization imaging. 22,23 A similar technique, the reflection contrast microscopy, has frequently been used for detection and quantification of the peroxidase product diaminobenzidine (DAB), 24-27 but requires the use and disposal of the toxic substrate DAB. In bright-field microscopy, absorbance techniques can be applied without expensive microscopic equipment as is usually required for fluorescent techniques. Absorbance techniques, which measure the optical density of enzymatic reaction products, have been described for the use of peroxidase- and alkaline phosphatase-linked detection systems. 5,28 Absorbance measurement of the peroxidase substrate DAB or the alkaline phosphatase substrate Fast Red has been used in several approaches to determine antigen concentrations. 29-33 In addition, several approaches of standardization have been made to prove a quantitative correlation of staining intensity and antigen or antibody concentration. 34-39 Independent of the method used, standardization of the technical procedure is crucial for obtaining accurate and reliable results. Moreover, standardization is needed to meet the requirements of quality control concerning objectivity and reproducibility. 6,28,35,40 An additional prerequisite in diagnostic pathology is the necessity for stability of staining and permanent mounting for long-term storage of sections to provide documentation and verification of the diagnosis. Here, we present the use of the substrate Vector Red for alkaline phosphatase-linked immunostaining and microdensitometry. Vector Red provides a stable bright red precipitate visible in bright-field and fluorescence microscopy, and it offers the possibility of permanent mounting and long-term storage. In addition, we compared different detection techniques (epipolarization, fluorescence, and absorbance) for quantitative evaluation of staining intensity. Standards for both immunogold- and alkaline-phosphatase-based techniques were created by means of agarose blocks into which antibodies coupled with either ultra-small gold particles or alkaline phosphatase were incorporated. The linearity of staining intensity was characterized by using different antibody concentrations, sections of different thickness, and increasing development time. In essence, the amount of precipitate or dye (silver grains or Vector Red) was found to be linear with antibody concentration, thickness of sections, and development time, thus qualifying both detection methods as appropriate for quantitative analysis of light-microscopic immunohistochemistry, offering several advantages in comparison with common staining and detection techniques. | |||||||||||||||||
Materials and Methods Reagents Agarose low electroendosmosis was obtained from Boehringer Mannheim (Mannheim, Germany). Chicken egg white was provided from Chroma (Münster, Germany), bovine serum albumin, poly-l-lysine, methyl green, nuclear Fast Red, Mayer’s hematoxylin, the alkaline phosphatase substrate Fast Red and 4′, 6-diamidino-2-phenylindole (DAPI) were obtained from Sigma (Deisenhofen, Germany). Paraffin liquid (no. 107162) and low-temperature paraffin (no. 107150) were both obtained from Merck (Darmstadt, Germany).Anti-CD45 antibody (no. MCA-43G, clone MRC OX-1) was purchased from Serotec (Oxford, UK). Secondary antibodies goat-anti-mouse (alkaline phospatase-conjugate, Texas Red-conjugate, ultra-small gold-conjugate) were provided by Nanoprobes (Stony Brook, NY) and Biotrend (Cologne, Germany). The silver enhancement solution was acquired from Aurion (Wageningen, The Netherlands). The alkaline-phosphatase substrate kit Vector Red was purchased from Vector Laboratories (Burlingame, CA). Clarion and Crystallmount were purchased from Natutec (Frankfurt, Germany), and Permafluor was provided by Immunotech (Hamburg, Germany). Preparation of Standard Blocks Agarose LE was dissolved in boiling double-distilled water resulting in a 4% agarose solution. During constant stirring, the solution was cooled down to 42°C. Meanwhile, the antibody was admixed in different concentrations (AP-conjugated antibody 1:125, 1:250, 1:500, colloidal gold-conjugated antibody 1:50, 1:100, 1:200, 1:400) to a solution of 5% bovine serum albumin and 10% egg white in double-distilled water. The antibody-containing solution was then added 1:1 to the agarose solution and thoroughly mixed. Subsequently, the mixture was aspirated in a 1-ml syringe and rapidly cooled down at 4°C to accelerate gelling of the agarose. The solid standard block was removed from the syringe and cut into 3- to 5-mm-sized cylinders.Paraffin-embedding was performed by dehydration of the blocks in a graded series of alcohol and immersion in a mixture of 6:4 low-temperature paraffin and paraffin oil at 40°C overnight. Sectioning of 5, 10, 15, and 20 μm was performed in a cryostat (CM 3050; Leica, Benzheim, Germany) at −25°C. Sections were placed in cold water and mounted on poly-l-lysine and chicken egg white-coated glass slides. Staining of Standard Sections The sections were dewaxed, rehydrated, and washed in double-distilled water for 5 minutes and sections with incorporated AP-labeled antibody were incubated with Vector Red substrate for different periods (10 minutes to 60 minutes at intervals of 10 minutes) in a humid chamber. For development the slides were placed in the dark, at a constant temperature of 20°C in an incubator (Heraeus Instruments GmbH, Hanau, Germany). For bleaching experiments sections of the AP-labeled standard blocks were additionally developed with the AP-substrate Fast Red for 40 minutes.Sections of immunogold standards were incubated with silver enhancement solution for 40 minutes under constant conditions in the same incubator. The incubation was then rapidly interrupted by thoroughly rinsing the sections in double-distilled water. Negative staining controls for background subtraction were made without incubation of substrate solution or silver enhancement solution. Subsequently, the sections were dehydrated and permanently mounted. Animal Preparation The rats (male; body weight, 350 to 400 g) were deeply anesthetized with sodium pentobarbital (100 mg/kg body weight intraperitoneally). After local anesthesia with 2% xylocaine and median incision, the trachea was dissected and a tracheal cannula was immediately inserted. A median laparotomy was performed and subsequently the rats were anticoagulated with 1,000 U of heparin. After midsternal thoracotomy, the right ventricle was incised, a cannula was fixed in the pulmonary artery, and the apex of the heart was cut off to allow pulmonary venous outflow. Simultaneously, pulsatile perfusion with buffer solution was started. The buffer contained 2.4 mmol/L CaCl2, 1.3 mmol/L MgCl2, 4.3 mmol/L KCl, 1.1 mmol/L KH2PO4, 125.0 mmol/L NaCl, 25 mmol/L NaHCO3 as well as 13.32 mmol/L glucose (pH ranged between 7.35 and 7.40). The lungs were continuously perfused for ~5 minutes for washout of blood.Tissue Preparation Lung tissue was chosen as test tissue for this study, because its high content of elastic fibers makes it a difficult object for evaluation, eg, in fluorescence microscopy. Fixation was performed by tracheal instillation of a 3% paraformaldehyde solution at a pressure of 20 cm H2O and immersion of the lungs in the same fixation solution for 1 hour (n = 5, each group). For paraffin embedding the entire lung was dissected into tissue blocks from all lobes. The tissue blocks were embedded in low-temperature paraffin with a melting temperature of 40 to 42°C. Sectioning of 10 μm thickness was performed from all paraffin blocks on a paraffin microtome (Reichert-Jung Histoslide 2000; Cambridge Instruments, Nussloch, Germany).In addition lungs were instilled with TissueTek OCT compound and snap-frozen in liquid nitrogen, dissected in tissue blocks from all lobes, and stored at −80°C. Sectioning of 10 μm thickness was performed from frozen tissue blocks of both left and right lungs in a cryostat (CM 3050; Leica, Benzheim, Germany). Immunohistochemistry Immunogold-Silver Staining The paraffin sections were dewaxed, rehydrated, and washed in phosphate-buffered saline (PBS) (0.01 mol/L, 150 mmol/L NaCl, pH 7.6) for 3 × 5 minutes. Cryosections were fixed in a 3% paraformaldehyde solution for 5 minutes and washed in PBS 3 × 5 minutes. The sections were pre-incubated in PBS containing 5% goat serum, 1% bovine serum albumin (BSA), 0.1% BSA-C, 0.05% Tween-20, and 0.02 mol/L glycine to block nonspecific binding. Overnight incubation with the monoclonal anti-CD45 antibody diluted 1:100 in PBS containing 1% BSA, 0.1% BSA-C, and 0.05% Tween-20, was performed at 4°C. The sections were then washed in PBS and incubated with the secondary gold-conjugated antibody diluted 1:400 overnight at 4°C. Next the sections were washed in PBS again and fixed for 5 minutes in 2% phosphate-buffered glutardialdehyde. After several washes in glass-double-distilled water the sections were incubated in Silver Enhancer solution for 40 minutes. Counterstaining of the sections was performed with nuclear Fast Red. Alkaline Phosphatase Staining Paraffin and cryosections were processed accordingly. The sections were pre-incubated in PBS containing 5% goat serum, 1% BSA, and 0.05% Tween-20 to block nonspecific binding. Incubation conditions with the primary anti-CD45 antibody (1:100 in PBS containing 1% BSA and 0.05% Tween-20) were as described above. The secondary AP-conjugated antibody (dilution, 1:4,000) was incubated in the same dilution buffer overnight at 4°C. Afterward, washing of the sections in PBS for 3 × 5 minutes was performed. Subsequently, sections were developed with a Vector Red Substrate Kit for 30 minutes. Levamisol, 2.5 mmol/L, was added to inhibit endogenous AP activity. Counterstaining of these sections was performed with methyl green. Texas Red Staining Cryosections were fixed in a 3% paraformaldehyde solution for 5 minutes and washed in PBS for 3 × 5 minutes. The sections were preincubated and treated with primary antibody as described in Alkaline Phosphatase Staining. The secondary Texas Red-conjugated antibody was applied in a concentration of 1:1,000 overnight at 4°C. Counterstaining was performed with Mayer’s hematoxylin or DAPI. Control staining was performed by omission of the primary antibody and substitution with nonspecific serum at the same dilution. Mounting Immunogold and AP-Vector Red-stained sections were permanently mounted with Clarion and coverslips, whereas Texas Red-labeled sections were mounted in an aqueous mounting medium (Permafluor). AP-Fast Red-stained sections were mounted without coverslips with the water-soluble Cyrstalmount, which hardens like a permanent mounting medium and can be visualized with oil immersion. Image Analysis An image analysis system with a 12-bit cooled charge-coupled device camera (Sensys KAF 1400; Photometrics, Tucson, AZ) mounted on a fully automated Leica DM RXA (Leica, Wetzlar, Germany) was used to digitize gray-scale images to a Dual-Pentium 200 MHZ host computer. The 12-bit camera has a high dynamic range with a nonlinearity lower than 0.5%. Microscope settings were kept constant throughout all measurements (objective, 40× oil; Leica PL Fluotar 40×/0.75). For epipolarization and fluorescence imaging a stabilized 12-V high-pressure mercury-vapor lamp, 100 W, was used for illumination. A Leica IGS epipolarization filterblock (POL no. 513813) and a Texas Red filterblock (TX2 no. 513843) were used for either epipolarization or fluorescence microscopy. Absorbance measurement was performed with a custom-designed filter for absorbance of the substrate Vector Red (central wavelength, 525 nm; half band width, 10 nm ± 2 nm) in optical quality (manufactured by Omega Optical, Inc., Brattleboro, VT). The optimal central wavelength was determined by measurement of the substrate in a Leica MPV SP Microscope Photometer System by courtesy of Leica. Estimation of section thickness by detection of autofluorescence was performed with a fluorescein isothiocyanate filter. A stabilized 12-V tungsten-halogen lamp, 100 W, was used for illumination. Bright-field images were taken with a Leaf Microlumina (Scitex Corporation Ltd., Herzlia B, Israel), mounted on the 35-mm photo exit of the Leica DM RXA. Adjustment of all microscope settings was stored and recalled before measurement. Calibration of the measurement system with a reference slide was done before measurement. Gray-scale images were digitized to 12 bit accuracy resulting in an intensity scale ranging from 0 to 4095. Image analysis was performed by means of the image analysis program ImagePro 3.0 (Media Cybernetics, Del Mar, CA). For direct visualization of staining intensity a pseudocolor scale with 11 colors was chosen, each representing an equal sector of the intensity scale, and applied to the images. Background measurement was performed to evaluate the influence of nonspecific antibody binding. Measurement Procedure From the standard blocks at least three sections of each thickness (5, 10, 15, and 20 μm) and each staining time (intervals of 10 minutes) were measured (gain 1, exposure time: Vector Red, 400 ms; IGSS, 20 ms). From each section 5 images (1024 × 1024 pixels) were digitized. Data of integrated optical density and area of each image were automatically measured and subsequently transferred into the spreadsheet program EXCEL 7.0a (Microsoft Corporation, Redmond, WA). CD45-immunostained alveolar macrophages were likewise selected in an area of interest and automatic measurement was performed from 5 to 10 sections of each different immunostaining technique. For investigation of photobleaching of Vector Red, Fast Red, Texas Red, and immunogold-silver in absorbance, epipolarization, or fluorescence detection intensities, images from at least three different areas per section were digitized and intensity was measured. At certain intervals of permanent light exposure (every 0.5 minute during the first 10 minutes, after 15, 30, and 60 minutes, after 24 hours) the signal intensities were measured. Autofluorescence for quality control of section thickness was measured from eight randomly chosen regions of at least three unstained sections. Detection of the autofluorescence signal was performed at 480 ± 40 nm excitation and 527 ± 30 emission (fluorescein isothiocyanate filter, L5 no. 513840; Leica). The autofluorescence signal was rather low, so the detection had to be optimized with higher camera gain (gain 3) and exposure time (8 seconds). Statistical Analysis Analysis of variance was used to evaluate differences among different groups. A value of P < 0.05 was considered significant. Data are given as mean ± SEM. | |||||||||||||||||
Results Segmentation Segmentation of signal in immunogold-silver staining was achieved by epipolarization microscopy using a special epipolarization filter. Epipolarization depiction of the silver-enhanced colloidal gold particles created a complete segmentation between positively stained and nonstained tissue.The individual transmission spectra of the substrates Vector Red, and the counterstains methyl green and Mayer’s hematoxylin are shown in Figure 1
In fluorescence microscopy both Vector Red and Texas Red staining were detected at >615 nm. Segmentation was, however, limited by autofluorescence of the tissue, which may be rather strong in a variety of organs, eg, lung tissue because of the abundance of elastic fibers. Standard Agarose Blocks Embedding of agarose blocks in paraffin and lowering of the temperature were necessary for precise sectioning of the standard blocks. Accuracy of section thickness was assessed by measuring the extent of autofluorescence, to which it is tightly correlated. 41 In addition, to minimize the impact of section thickness on measurement accuracy at least three sections for each measurement were used. 42 The antibodies were found to be homogeneously distributed within the agar matrix (Figure 2) ![[triangle]](corehtml/pmc/pmcents/rtrif.gif) . Silver-enhancement of colloidal gold occurs by deposition of metallic silver, which clusters around colloidal gold particles and produces a grain-like appearance (Figure 2A) . Staining intensity, however, was determined by integration of staining intensity over a complete microscopic field of 36,503.62 μm2. Alkaline-phosphatase-related staining in contrast resulted in a homogeneous distribution with equal intensity over the complete field (Figure 2B , inverted image).
The linear range of both absorbance and fluorescence of Vector Red was assessed throughout a development period of 60 minutes (Figure 3)
Absorbance measurement was linearly related to different antibody concentrations admixed to the agar blocks (Figure 4A)
Stability of Staining The stability of the staining intensity at light exposure was examined throughout a 1 hour period (Figure 6) and after 24 hours. A considerable bleaching of the staining was noted for the fluorescent dye Texas Red (Figure 6) , but was also detected to a minor extent with the alkaline phosphatase substrates Vector Red and Fast Red in fluorescence microscopy (Figure 6 , Table 1 ). Absorbance measurement of Vector Red and Fast Red staining revealed stable intensities throughout a 1 hour period, but no significant change was observed throughout an observation period of 24 hours. Similarly, absorbance and epipolarization of immunogold-silver staining was entirely stable, without showing any fading throughout 24 hours (Figure 6) .
Background Development of alkaline phosphatase with Vector Red produces a bright-red precipitate, which is clearly detectable by absorbance with the described custom-designed filter. Nonspecific staining and background staining was low, both in paraffin-embedded tissue and cryosections. Staining was absent from sections where the primary antibody was omitted or nonspecific serum was applied. In contrast, fluorescence microscopy of Vector Red- and Texas Red-stained tissue is hampered by tissue-specific autofluorescence, which is extremely strong, eg, in lung tissue (Table 1) .Immunogold staining with subsequent silver enhancement showed excellent results in paraffin-embedded tissues. Positive-stained structures were unambiguously detectable and background staining was low. On use in cryosections, in contrast, immunogold-silver staining may exhibit nonspecific and background staining. For this reason, the segmentation of stained structures in epipolarization primarily depends on the antibody used. Application of epipolarization in pathological human tissue is limited by foreign bodies/material, eg, anthracotic pigment deposition in the lung, which shows epipolarization and may hinder segmentation and determination of positively stained structures (Table 1) Application CD45 immunostaining was performed on differently embedded tissue and with different immunostaining protocols. Immunogold-silver staining (Figure 7, A–C) resulted in a strong and clear signal, which showed good contrast to the counterstain nuclear Fast Red in bright-field microscopy (Figure 1A) . Epipolarization created a complete segmentation between stained and unstained structures (Figure 7B) . Different staining intensities can be visualized using a pseudocolor depiction, which represents different gray-scale ranges (Figure 7C) . Vector Red staining gives best contrast with methyl green counterstaining (Figure 7D) , which is not visible using the custom-designed absorbance filter (Figure 7E) .
Texas Red immunofluorescence gives sufficient staining results (Figure 7G) | |||||||||||||||||
Discussion During the past few years, computer-assisted image analysis has been increasingly used for quantitative evaluation of histopathological and cytopathological features in the research areas and in diagnostic pathology. 1,3,40 The quantification of the final reaction product of immunohistochemistry has been addressed in numerous studies, and different staining techniques were evaluated with respect to standardization and linearity, prerequisites for obtaining reliable and reproducible quantitative results. 5,28,39 Such standardization also includes technical steps, therefore the influence of sectioning, antibody concentration, and staining time were evaluated in this study. To perform a comparison of different methods in quantitative microdensitometry, we created artificial test blocks by incorporation of antibodies labeled with either immunogold or alkaline phosphatase into agarose, and this standard specimen served for characterization of the immunogold-silver and Vector Red-staining intensity. There have been previous efforts to create artificial test standards for the characterization of reaction products, eg, the peroxidase substrate DAB, by incorporation of antigens or antibodies in agarose or gelatin media or coupling them with Sepharose beads. 32,34,43 Linearity between DAB absorbance and concentration or staining time and therefore suitability of DAB for densitometric quantitation was demonstrated in all studies. 32,34,43 DAB deposition can also be quantified using reflection contrast microscopy, 26,29 an epi-illumination technique that detects the reflected light from metal particles. 24 DAB is, however, a highly toxic substance and has thus to be handled with particular care and requires special disposal; it was therefore not considered to be appropriate for routine diagnostic immunostaining. Antibody-linked immunogold particles can be clearly visualized light microscopically after amplification by silver enhancement and use of epipolarization microscopy technique. 16,20,21,44 The combination of immunogold-silver staining and epipolarization microscopy is a highly sensitive method applicable for both immunohistochemistry and in situ hybridization, producing excellent segmentation of the bright-appearing immunopositive signal as shown in the current and previous studies. 22,23,45,46 Image analysis of our standard blocks, which resemble tissue samples with a defined concentration of label, demonstrated a linear relationship between epipolarization intensity and antibody concentration, as was anticipated from a on-slide enzyme-linked immunosorbent assay technique combined with epipolarization microscopy. 20 In addition, unrestricted light stability throughout time and a spatially precise signal with no out-of-focus signal recommends the immunogold-silver technique for quantitative studies with evaluation of staining intensity. Measurement of CD45 immunogold-staining intensity of the lung tissue with epipolarization technique showed the lowest scattering of data of all methods presently investigated. High sensitivity, excellent segmentation, sharp contrast in the bright-field in combination with nuclear counterstains like nuclear Fast Red or methyl green, and the possibility of permanent mounting and long-term storage are further advantages of this technique. However, in cryosections immunogold-silver staining produces more background than in paraffin sections, which limits the applicability for quantitative evaluation. Moreover, when assessing pathology samples, the frequent occurrence of foreign body material, such as anthracotic pigmentation in human lung tissue, represents a problem for the segmentation of the immunostaining signal even in epipolarization microscopy. An alternative for use in paraffin- and cryosections, with no limitations concerning the appearance of foreign body material, is the alkaline phosphatase-based immunostaining with development of different colored substrates. Quantitative evaluation of alkaline phosphatase-based immunohistochemistry has been reported in a limited number of studies, using the substrate Fast Red 29,37,38 and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. 36 Fast Red was recommended for use in double-labeling studies with fluorescence. 47-49 Vector Red displays nearly identical features as Fast Red, including bright red color in the bright-field and intense fluorescence detectable in rhodamine or Texas Red filter systems. In the present study, linearity of Vector Red absorbance and amount of incorporated enzyme, development time, or section thickness were shown, demonstrating suitability of this tool for quantitative assessment. Control of the section thickness was performed by measurement of fluorescein-isothiocyanate autofluorescence, as previously described. 41,50 Depending on the required accuracy of measurement a correction of section thickness may additionally be considered by applying a correction formula. 1 The section thickness may be measured either by autofluorescence intensity or immunohistochemical background staining. 1 Comparison between Vector Red absorbance and fluorescence detection revealed that the linear range for absorbance measurement comprised at least 60 minutes of development time, whereas fluorescence of Vector Red was only linear between 20 and 40 minutes of development time. Measurement of absorbance is thus less critical than fluorescence when the development time has to be adjusted for optimal immunohistochemistry. An important advantage of Vector Red is the absolute light stability in absorbance measurements. Both Vector Red and Fast Red show considerable photobleaching when exposed to mercury light in fluorescence microscopy (this study and Speel et al 48 ) which is, however, much lower and delayed compared to commonly used fluorescent labels like Texas Red, as demonstrated in the current study. Because of photobleaching, fluorescence measurements result in higher variability of data as compared to absorbance measurements. However, fluorescent measurements can be reliably performed when taking this fact into consideration, eg, by using an automated microscope with a light shutter to reduce the time of light exposure to a minimum. 51,52 Sections stained with common fluorescent markers or Fast Red have to be mounted in aqueous mounting medium for fluorescence microscopy, which hinders storage and long-term stability. Vector Red-stained specimens, in contrast, can be permanently mounted with coverslips and be conserved for months or years without loss of staining intensity. Good results were obtained in the present study with the water-soluble mounting medium Crystalmount, eg, for mounting of Fast Red-stained specimens, which hardens like any other permanent mounting medium and provides good optical quality when covered with immersion oil. The counterstaining of Vector Red staining has to be selected carefully if the slides are to be observed in the bright-field and fluorescence microscopy, because common nuclear stains used in bright-field microscopy show intense fluorescence. In fluorescence microscopy of Vector Red-stained structures, counterstaining with methyl green was found to be inappropriate, because of the extremely strong fluorescence of methyl green throughout almost the entire fluorescent spectrum. Mayer’s hematoxylin, in contrast, was suitable for use in bright-field and fluorescence microscopy because segmentation of the immunostaining was possible. In contrast to Mayer’s hematoxylin, other commonly used hematoxylin counterstains again display intense fluorescence. As expected, DAPI is suitable for counterstaining in fluorescence microscopy, but is unfortunately not useful for bright-field microscopy. For quantitative evaluation with absorbance measurement, we found methyl green to be the ideal counterstain, because transmission spectra of Vector Red and methyl green do not overlap and are thus easy to separate with appropriate filters. For Fast Red staining, with either bright-field or fluorescence microscopy, the choice of counterstain is limited to water-soluble dyes, and thus Mayer’s hematoxylin is a suitable nuclear stain for this purpose. Additional problems in using fluorescence microscopy for quantitative evaluation of staining intensity may arise from tissue-specific autofluorescence and out-of-focus fluorescence, which both influence accuracy of measurements. Autofluorescence is particularly disturbing in lung tissue, because of the high content of elastic fibers within the alveolar septum and in pulmonary vessels, which may hinder segmentation of the fluorescence signal. When measuring epipolarization signals of immunogold-silver deposits an interference by background signals with the immunopositive signal does not occur because silver-based immunohistochemistry is not influenced by the presence of endogenous enzymatic activity and thus exact segmentation of the signal is possible. 53 It is important to mention that the described methods of quantification do not yield absolute quantitative data, but show relative changes of protein expression in different samples. Prerequisite for obtaining reliable and reproducible results are equal conditions of tissue processing, which requires standardization and calibration of each step of tissue processing. This includes parameters such as period of time until fixation starts, type of fixative and length of fixation, size of tissue samples, storage conditions, and parameters of tissue embedding. For each different antigen, which is to be localized and measured, the individual staining conditions again have to be optimized. Variations between antigens such as the amount of antigen, stability, or metabolic break down determine which technique of tissue processing may be used: paraffin-embedded tissue versus frozen tissue, necessity of antigen retrieval, or variable staining conditions (ie, type and concentration of antibodies, incubation periods). In experimental applications the conditions of tissue processing can more easily be held equal than in clinical applications. If certain parameters of processing clinical tissue samples cannot be fully standardized, at least the influence of these parameters on the immunostaining should be analyzed. The grade of standardization influences the accuracy of the quantitation method, which means that low standardization results in high variability of measurement and thus differences between groups have to be high to be detected. The grade of standardization, which is required for accurate measurement, in the last instance depends on the individual antigen, its expression, stability, and the differences of variation expected to be measured in different tissue samples. If sufficient standardization, calibration, and proper controls for quantitation are ensured, quantitative microscopy can be a helpful tool in experimental and diagnostic pathology. 1,39,40 In conclusion, microdensitometry of Vector Red absorbance is a suitable method for quantification of staining intensity and meets current requirements for a stable, reproducible, and economical technique to be applied in diagnostic pathology. Applicability in paraffin-embedded tissue as well as in cryosections, excellent segmentation, linearity over a wide range, light stability, and feasibility for permanent mounting and for long-term storage are the outstanding features of this approach. Absorbance measurement does not require expensive equipment and can be performed with a common light microscope, an appropriate absorbance filter, and a stabilized transformer for steady illumination. In comparison with other staining and measurement techniques reported in the literature and those presently investigated in parallel, we consider microdensitometry of Vector Red absorbance as being the most suitable approach for standardized quantitative evaluation. | |||||||||||||||||
Acknowledgments We thank G. Müller for excellent technical assistance; and Dr. R. L. Snipes, Department of Anatomy, Giessen, for linguistically reviewing the manuscript. | |||||||||||||||||
Footnotes Address reprint requests to Leander Ermert, M.D., Institut fuer Anatomie und Zellbiologie, Aulweg 123, 35385 Giessen, Germany. E-mail: leander.ermert/at/anatomie.med.uni-giessen.de. Supported by the Deutsche Forschungsgemeinschaft (SFB 547 “Kardiopulmonales Gefäβsystem”). | |||||||||||||||||
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