TECHNICAL NOTE |
Correspondence to: Brian G. Short, Senior Pathologist, SmithKline Beecham Pharm., 709 Swedeland Rd., UE0364, King of Prussia, PA 19406-0939.
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Summary |
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Immunohistochemical markers for proliferation (bromodeoxyuridine, BrdU) and apoptosis (in situ terminal deoxynucleotide transferase dUTP nick end-labeling, TUNEL) were localized within glutathione S-transferase (GSTP)-positive hepatic foci in rats. Using the TechMate Automated Staining System (BioTek Solutions; Santa Barbara, CA), formalin-fixed, paraffin-embedded sections were run through a double-label avidin-biotin-immunoperoxidase protocol in less than 10 hr. Steam heat-induced epitope retrieval and/or proteolytic digestion preceded each labeling procedure. Color development was achieved using diaminobenzidine (DAB) with nickel enhancement for BrdU and TUNEL and VIP for GSTP. Results illustrate clear staining, brown-black BrdU-positive nuclei or TUNEL-positive apo-ptotic bodies within purple GSTP-positive hepatocytes. This automated procedure provides a method to easily identify and quantitate proliferating or apoptotic cells within foci of altered hepatocytes in rat liver and may have general applications for studies of cell or tissue kinetics during development, differentiation, and various pathological conditions in animals and humans. (J Histochem Cytochem 45:1299-1305, 1997)
Key Words: automation, proliferation, apoptosis, bromodeoxyuridine, TUNEL, steam heat, glutathione S-transferase, foci, rat
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Introduction |
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Double immunohistochemical labeling techniques are popular because their ability to demonstrate two antigens within the same piece of tissue has many applications in diagnosis and research (
The control of cell numbers in tissues or specific cell populations is highly dependent on the balance between cell birth and cell death, or apoptosis (
Methods for detecting cell proliferation in rodent liver have been reviewed (
There are several methods for detecting apoptosis in rat hepatocytes (
Our objective was to develop an automated double labeling technique to detect both proliferating and apoptotic cells in GSTP+ hepatic foci. BrdU- or TUNEL-labeled focal (GSTP+) and nonfocal (GSTP-) hepatocytes of rats were identified and quantitated following an initiation-promotion protocol. This automated double labeling technique provides advantages of minimal effort in an accurate, consistent, and expedient manner and may have applications for studies of proliferation or apoptosis within a wide variety of tissues and cell types.
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Materials and Methods |
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Animal Treatment, Tissue, and Slide Preparation
In an initiation-promotion protocol, eight male Sprague-Dawley rats were given 150 mg/kg diethylnitrosamine (Sigma Chemical; St Louis, MO) by IP injection, followed 2 weeks later by an 8-week period of exposure to a proprietary rat hepatocarcinogen. During the last week of treatment, BrdU (30 mg/ml; Sigma) in PBS was delivered by osmotic pump infusion (2ML1, 10 µl/hr; ALZA, Palo Alto, CA). Rats were sacrificed by carbon dioxide asphyxiation and exsanguination. Livers were collected and fixed in 10% neutral buffered formalin (NBF) for 3 days and processed through graded alcohols and toluene to paraffin. Paraffin-embedded liver was cut into 5-µm-thick sections, placed on Capillary Gap Plus Microscope slides (BioTek Solutions; Santa Barbara, CA), and air-dried. Slides were deparaffinzed in xylene, rehydrated, and loaded into the TechMate slide holder.
First Label: BrdU or TUNEL
BrdU.
Reagents for this assay, excluding the primary antibody, were purchased as a kit (BioTek Solutions). Slides were steamed (Handy Streamer Plus; Black and Decker, Shelton, CT) for 20 min in heat-induced epitope retrieval (HIER) buffer. The HIER procedure was performed manually but automation of this technique has been described (
TUNEL. ISEL using the ApopTag detection kit (S7100 kit; Oncor) was used to detect cells undergoing apoptosis. Sections were processed in accordance with kit instructions, with modifications as noted below. The TechMate system automated protocol began with a 15-min incubation in 3% H2O2 in buffer, followed by a 10-min proteolytic digestion in prediluted proteinase K. Slides were incubated in equilibration buffer for 2 min, followed by incubation in working strength terminal deoxynucleotidyl transferase (TdT) enzyme at 37C for 70 min. The TdT working strength enzyme consists of TdT enzyme and digoxigenin-11-dUTP for end extension of the 3'OH ends of double- or single-stranded DNA of fragmented DNA. This was followed by stop/wash and anti-digoxigenin-peroxidase steps, which were identical to kit instructions. Color development with DAB and nickel chloride was identical to that described with BrdU, as above.
Second Label: GSTP
Reagents for this assay, excluding the primary antibody and the VIP chromogen (Vector Labs; Burlingame, CA) were purchased as a kit (BioTek Solutions). After automated staining of the first label, slides were removed from the TechMate machine and steamed manually in HIER for 20 min before automated staining for GSTP. Residual biotin from the previous BrdU staining procedure was blocked using an avidin-biotin blocking kit (Vector Labs) and peroxidase used in the first procedure was blocked with 3% H2O2 for 15 min. Slides were incubated in rabbit anti-human GSTP (Dako) in 1~100 dilution in PBS buffer for 45 min, followed by biotinylated anti-rabbit and ABC-peroxidase for 30 min each. Slides were developed using the VIP chromogen for 3-10 min. The VIP chromogen development was visually monitored until desired staining density was achieved. Slides were counterstained with hematoxylin, dehydrated, cleared, and coverslipped using a xylene-based mounting medium.
Controls
Positive controls included collection, sectioning, and staining for BrdU or TUNEL incorporation in duodenum along with liver in the same slide from each rat. Additional tissue slides were stained separately for BrdU, TUNEL, or GSTP to verify that staining sensitivity was not decreased by double labeling. Negative controls for BrdU or GSTP immunohistochemical staining included slides that were incubated with normal mouse or rabbit serum in place of BrdU or GSTP primary antibodies, respectively. To provide a negative control for TUNEL, we omitted TdT from the labeling mix. Specificity was controlled by comparing TUNEL with morphology as observed in H&E-stained slides.
Quantitation of Proliferation and Apoptosis
An electronic imaging program (Image Pro Plus; Media Cybernetics, Silver Springs, MD) was used as an aid in counting by projecting the electronic image onto a monitor for identification and tagging of BrdU- or TUNEL-positive hepa-tocytes. BrdU and TUNEL labeling indices of nonfocal (GSTP-) hepatocytes were determined by dividing the number of BrdU-positive hepatocyte nuclei or TUNEL-positive hepatocytes, respectively, by the total number of hepatocytes scored and multiplying by 100. A minimum of 1500 or 4000 hepatocytes were scored in random fields for BrdU or TUNEL labeling, respectively, from the left liver lobe of eight rats. BrdU and TUNEL labeling indices of focal (GSTP+) hepatocytes were determined by dividing the number of BrdU-positive hepatocyte nuclei or TUNEL-positive hepatocytes, respectively, by the total number of hepatocytes within each GSTP+ focus and multiplying by 100. BrdU and TUNEL labeling indices were determined for 20 random hepatic foci per section and were averaged to determine the focal BrdU or TUNEL labeling index for each rat. Mean ± SEM focal and nonfocal BrdU and TUNEL labeling indices were calculated. Statistical analysis was conducted to determine significant differences between focal and nonfocal labeling indices. Focal and nonfocal labeling indices were compared by a Student's t-test if the data passed a test for normality (ANOVA) or Mann-Whitney Rank Sum test if the data failed the normality test.
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Results |
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The combination of dark brown-black DAB staining of BrdU or TUNEL and the purple VIP chromagen staining for GSTP gave excellent contrast and staining quality (Figure 1A and Figure 1B). BrdU-labeled nuclei of normal hepatocytes were easily distinguished from labeled nonhepatocytes by their large, spherical contour and their central cellular location. Staining intensity of GSTP+ hepatocytes varied throughout each section, which is consistent with the variable phenotypic expression of this marker within rat hepatic foci. BrdU-labeled hepatocyte nuclei were easily identified within purple GSTP+ hepatocytes and were readily distinguished from unlabeled hepatocyte nuclei within the same focus.
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We first examined H&E-stained liver sections for apoptosis because the current standard for recognition and quantitation of apoptosis in tissue sections is based on morphological features observed with this stain (
In TUNEL/GSTP double labeled slides, apoptotic cells and bodies stained dark brown and hepatic foci stained purple (Figure 3A and Figure 3B), which enabled easy identification of various stages of apoptosis. TUNEL-labeled apoptotic bodies were observed as discrete, membrane-bound, chromatin- or nonchromatin-containing bodies of variable size within the cytoplasm of GSTP+ hepatocytes (Figure 3B) or as diffuse light or dark brown cytoplasmic staining that covered the entire cell (Figure 3B). Occasional bodies did not stain positive with TUNEL (Figure 3B). TUNEL-labeled apoptotic cells, characterized as brown staining confined to the nucleus of intact hepatocytes, were occasionally observed (Figure 3B) but were less numerous than apoptotic bodies.
Positive control slides of duodenum demonstrated dark brown BrdU-labeled cells that covered the entire surface of the intestinal villi or dark brown TUNEL-labeled cells at the tip of the villi. The pattern of intestinal BrdU or TUNEL labeling was consistent with that described with this tissue after continuous (>4 days) BrdU labeling (deFazio et al. 1987) or TUNEL labeling (Gavrielli et al. 1992). Liver slides of single labeling of BrdU, TUNEL, or GSTP verified that the double labeling technique did not decrease binding of either antibody because staining intensity for BrdU, TUNEL, or GSTP was similar between single and double labeling procedures. Staining of BrdU and GSTP control slides incubated with no primary antibody was negative. Omission of TdT from the TUNEL mix completely abolished labeling.
Quantitation of BrdU and TUNEL labeling indices of nonfocal and focal (GSTP+) hepatocytes is presented in Table 1. Proliferation and apoptosis within focal hepatocytes were increased approximately fourfold greater than nonfocal hepatocytes, indicating the utility of demonstrating differences in proliferation and apoptosis between focal and nonfocal hepatocytes.
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Discussion |
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Double labeling of BrdU or TUNEL and cytoplasmic proteins in formalin-fixed, paraffin-embedded liver or other tissues from rats by manual methods has been described (
Use of steaming compared to microwave treatment for heat-induced epitope retrieval (HIER) is equally effective for most antigens (
The utility of an avidin-biotin blocking kit in our second label (GSTP) after HIER for 20 min is unknown, because the residual link and label system may have been deleted by HIER (
Results of this study demonstrate the utility of BrdU for detection of proliferating hepatocytes within GSTP+ foci. The advantage of BrdU is that it can be administered to animals for prolonged periods, increasing the cumulative total of cells undergoing DNA synthesis compared to that detected with endogenous markers, such as PCNA. Immunostaining for PCNA has the potential to identify cells in the G1, S, G2, or M phases of the cell cycle and can be used in archival tissue sections (
Apoptosis is best characterized by evidence of DNA fragmentation and morphological features by H&E, but these present serious drawbacks in quantitation (
Because DNA fragmentation is associated with various biological and artifactual situations, the ISEL technique is selective (rather than specific) for apo-ptotic nuclei (
Fewer TUNEL-labeled apoptotic cells were observed in this study compared to TUNEL-labeled apoptotic bodies. This was an expected finding, because the appearance of early apoptotic cells is limited to only a few minutes and apoptotic bodies are visible by light microscopy in the liver for a few hours (
Diffuse cytoplasmic TUNEL staining observed in this study has been suggested to represent leakage of DNA fragments out of the nucleus of the phagocytized apoptotic body, either as part of the apoptotic process or as an artifact of the subsequent tissue processing and staining procedure (
The automated BrdU/GSTP and TUNEL/GSTP double labeling methods presented here produce material suitable for image analysis quantitation of proliferation and apoptosis within rat hepatic foci. Automating the quantitation of BrdU- or TUNEL-labeled cells in normal hepatocytes from rat and mouse liver is described, as well as image analysis of hepatocytes expressing GSTP (
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Acknowledgments |
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We thank Thomas Covatta for assistance in preparation of the photomicrographs.
Received for publication November 13, 1996; accepted April 8, 1997.
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