ARTICLE |
Correspondence to: James J. Liu, Vascular Biology Unit, Depts. of Cardiac Surgery and Medicine, U. of Melbourne Austin Hospital, Heidelberg VIC 3084, Australia.
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Summary |
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It has been difficult to quantify protein production in small pathological specimens by conventional techniques. We describe a new method for semiquantification of immunohistochemical staining, which involves application of the enzyme-labeled avidin (LAB) technique, coupled with an ultra-sensitive and fast chemiluminescent substrate for alkaline phosphatase. The entire procedure can be completed in less than 3 hr. The final step involves X-ray film exposure for 30 min, and the optical density of the subsequent images is examined with a microcomputer imaging device. The optical densities are translated into relative protein concentrations by a reference standard curve, obtained via an immunoblot. To establish a model for semiquantification of endothelial constitutive nitric oxide synthase (eNOS) protein, we compared the coronary arteries of WKY rats fed a normal chow diet to the coronary arteries of WKY rats fed a cholesterol diet. Using this technique, we have found a relative 130-fold decrease in eNOS in the cholesterol-fed group compared to the normal chow-fed group. (J Histochem Cytochem 46:257262, 1998)
Key Words: Nonradioactive, protein semiquantification, eNOS, immunohistochemistry, coronary artery, cholesterol diet, chemiluminescence
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Introduction |
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Endothelium-derived RELAXING FACTOR was first discovered by the work of
Quantification of protein in small pathological specimens is difficult with conventional methods. ELISA techniques or Western blotting often requires sample pooling to obtain a specimen sufficient for accurate analysis. The visual scoring system (H score) employs the ability of scientists to discriminate among immunohistochemical staining intensities (
We have developed a semiquantitative technique that can discriminate among antibody/antigen binding intensities. We have shown for the first time the difference between endothelial NOS binding intensities of hypercholesterolemic coronary arteries vs control coronary arteries. The method involves routine immunohistochemistry with a chemiluminescent final stage, which is detected by X-ray film. The resulting images on the X-ray film are processed with a microcomputer imaging device (MCID) and the optical densities of the images are recorded. The optical densities are then translated to relative protein concentration via a standard curve obtained with an immunoblot.
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Materials and Methods |
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Tissue Collection and Preparation
Ten 8-week-old WKY (normotensive) rats were divided into two groups of five. One group was fed a normal rat chow diet. The other group received a normal rat chow diet supplemented with 2% cholesterol (Sigma Chemical; St Louis, MO) and 8% peanut oil (GFW; Port Melbourne, Australia). After dieting for 15 weeks, rats were sacrificed and their hearts were excised and snap-frozen in a dry ice/isopentane bath. Specimens were kept at -70C until processed. A control rat heart and a cholesterol-fed rat heart were mounted on a single chuck. Three sections were cut and fixed per slide. If possible, tissues to be compared should be mounted on a single glass slide, so as to keep incubation and wash times constant.
Immunohistochemistry
Immunohistochemical staining of tissues was performed using a Dako Universal LSAB+ kit (Dako; Carpinteria, CA). Briefly, 8-µm tissue sections were cut with a cryostat and allowed to dry for 2 hr. A normal diet heart and a cholesterol diet heart were mounted on a single chuck and three sections were fixed per slide. This was repeated for each successive pair of hearts. The sections were fixed/dehydrated in acetone (Sigma) at room temperature (RT) for 10 min and allowed to dry. Once dried, the sections were placed in a plastic container with desiccant crystals and stored at -20C until needed. Sections were removed from -20C and allowed to equilibrate to RT before immunhistochemistry. Sections were rehydrated in 0.05 M Tris-HCl (Tris[hydroxymeth-yl]aminomethane hydrochloride) (Sigma), pH 7.4, for 5 min. Constitutive NOS antibody (Transduction Laboratories; Lexington, KY) and alkaline phosphatase (Sigma) were diluted in 0.05 M Tris-HCl, pH 7.4, containing 1% BSA (Sigma). A 20-min incubation in 20% goat serum (Dako; Carpinteria, CA) was used to decrease nonspecific binding. Sections were incubated with the eNOS antibody (0.1 µg/ml) or negative control antibody (Dako) (0.1 µg/ml) for 30 min and rinsed in a 0.05 M Tris-HCl, pH 7.4, bath with gentle rocking for 10 min. A biotinylated anti-mouse antibody supplied with the LSAB+ kit was used to incubate the tissue sections for 5 min and then the sections were rinsed in 0.05 M Tris-HC1, ph 7.4, in a gentle rocking manner for 10 min. The following 5-min incubation used a streptavidin molecule conjugated to alkaline phosphatase (Sigma) at a 1:100 dilution. Subsequently, a 10-min rinse in 0.05 M Tris-HCl, pH 7.4, in a gentle rocking manner was performed. The tissues were then equilibrated for 2 min in 100 mM Tris-HCl, pH 9.5, containing 100 mM NaCl. Incubation with disodium 4-chloro-3-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.13,7}decan}-4-yl) phenyl phosphate (CDP-Star substrate; BoehringerMannheim Biochemica, Mannheim, Germany) (diluted 1:100 in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl) for 5 min was performed to provide chemiluminescence. The substrate included 10 mM levamisole (Sigma) to inhibit endogenous alkaline phosphatases. The sections were placed in an oven at 20C to dry. The sections were then exposed for 30 min under Mamoray MR5 X-ray film (AGFA; Mortsel, Belgium). The following image was analyzed using an MCID.
Specimens were also processed using the LAB peroxidase method, according to the protocol provided in the LAB kit (
which was used to obtain the relative eNOS concentrations.
Microcomputer Imaging Device
The MCID was used to collect data in the following manner. The X-ray film was placed under the MCID camera and the corresponding microscope slide processed with the LAB-peroxidase method was placed under a microscope (low magnification, x 25). The camera height was adjusted to allow the image seen on the MCID monitor to be the same size as the image seen under the microscope. The relative light intensity on the MCID was adjusted to achieve a blue background and was increased until the negative control sections were not visible. The relative light intensity must not be changed during collection of data. The coronary arteries were then identified by scanning the microscope slide, and these arteries were assimilated to the coronary arteries seen with the MCID monitor. Once identified, the optical density of the endothelium was recorded by taking an outline of the endothelium.
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Results |
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The typical eNOS immunoreactivity of coronary arteries, as seen with the MCID, is shown in Figure 1. The sections stained with negative control antibody were visible only under lower relative light intensity, because higher relative light intensities reduced negative control image. The higher relative light intensity was used in our experiment. Immunoblotting techniques were used to establish the linearity of the antigen/antibody binding intensity (Figure 2). Regression statistics of the data revealed an inverse log relationship between binding optical density and antibody concentration (r = 0.99; p<0.0001, n = 3) (Figure 3). The formula
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The formula now reads
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Discussion |
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This article reports a novel method for immunohistochemical semiquantification. The method is simple, efficient, and rapid. Results are attainable in less than 3 hr. This method has vast potential applications, e.g., semiquantification of a protein in pathological tissues compared to normal tissues. We have also shown, for the first time, semiquantitative analysis of eNOS in coronary arteries of hypercholesterolemic rats.
Recently,
Time-course studies for primary antibody incubations should be set for each laboratory, because various antibodies require different incubation times. Previous experiments in our laboratory show a 30-min primary antibody incubation time to be optimal. Antibodyantigen saturation must be avoided if suitable comparison between samples is required (
Acetone was the fixative of choice because of its gentle fixative properties. Usually, paraformaldehyde is used in immunohistochemistry, although it is a harsher fixative. Although chemiluminescent Western blotting techniques are used routinely, localization of protein of interest is lost and ample specimen is needed for protein extraction and electrophoresis. Our method provides full localization of protein and the ability to use small amounts of specimen.
An increasingly recognized feature of vascular disease is an early and significant imbalance between endothelium-derived vasoactive agents. An equilibrium among endothelium-derived vasodilators and vasoconstrictors is essential for vascular homeostasis. Endothelium-dependent vascular relaxation is impaired in humans and animals with hypercholesterolemia, and this is believed to be an initial step in atherogenesis. Previously,
It is important to note that this method can report only the protein concentration relative to the standard curve obtained using the immunoblot. Purified eNOS protein used in the immunoblot might be structurally different to the eNOS protein found in tissue, and this could affect the antigen/antibody binding affinity. In addition, although incubation and wash times are consistent in both immunohistochemistry and immunoblotting, an immunoblot provides optimal conditions for antigen/antibody binding, whereas, in immunohistochemistry, tissue fixation may interfere with the permeability of the antibody or may directly affect the eNOS protein motif. Therefore, we stress the importance of reporting results with the use of the term "relative concentration" according to the immunoblot data. Unfortunately, not all proteins are available commercially, and if extraction and purification of a commercially unavailable protein cannot be considered, the antibody itself can be used instead of the protein in an immunoblot to create a standard curve. This is not as accurate, although the basic characteristics of the procedure can be established. Furthermore, it is not necessary to repeat the immunoblot on a daily basis as part of the protocol, once the characteristics of the system are established. Immunoblots in our laboratory are consistent on a day-to-day basis. However, we advise repetition of the immunoblot if the experiments are undertaken over an extended period of time, because phosphatase activity will decrease.
In summary, the protocol used is as follows:
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Quantitative immunohistochemistry |
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Immunoblot |
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In conclusion, we describe a novel semiquantitative immunohistochemical method for quantitation of protein in fresh frozen cryostat sections. The method involves the use of chemiluminesence and detection via X-ray film. The total procedure is completed in less than 3 hr, including X-ray film exposure time. An immunoblot is employed to create a standard curve, which is then used to translate optical density to relative protein concentration. The method is very sensitive, accurate, and rapid. Its potential applications are wide-ranging, because it is now possible to semiquantify protein production in small pathological specimens. This method provides a useful and convenient tool for quantitative studies in immunohistochemistry.
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Literature Cited |
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