ARTICLE |
Correspondence to: Ulrike Kämmerer, Dept. of Obstetrics and Gynecology, University of Würzburg, Josef-Schneider Str. 4, D-97080 Würzburg, Germany. E-mail: frak057@mail.uni-wuerzburg.de
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Summary |
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Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3'diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623630, 2001)
Key Words: immunohistochemistry, frozen section diagnosis, EnVision
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Introduction |
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SINCE 1941, when Coons and colleagues revolutionized the identification of tissue antigens using a direct fluoresence method (
A certain amount of information about rapid IHC techniques has been published (
A newly developed immunohistochemical detection system, EnVision, has recently become available and has been described as a very sensitive detection method for routine IHC (
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Materials and Methods |
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The EnVision (DAKO; Hamburg, Germany) used in this study is a two-step method in which application of the primary antibody is followed by a polymeric conjugate consisting of a large number of secondary antibodies (goat anti-mouse or goat anti-rabbit) bound directly to a dextran backbone containing HRP. One such conjugate contains up to 100 HRP molecules and up to 15 antibodies.
In this study, the results of immunostaining with a modified rapid protocol using EnVision on frozen sections were compared with the results of the HRP- and LSAB (labeled streptavidinbiotin; DAKO) methods routinely used for the immunostaining of frozen sections in the laboratory.
Tissue Specimens and Preparation
Tissue samples from surgical specimens (carcinoma of the breast, lymph nodes, including one with metastatic carcinoma, small intestine, skin, malignant melanoma, B-cell lymphoma infiltrating the breast, prostate, thyroid gland, and early pregnancy decidua) were snap-frozen in liquid nitrogen for 30 sec immediately after removal and then transferred to a cryostat (Leica; Bensheim, Germany). Serial frozen sections were cut at 5 µm and placed on APES (3-amino-propyltriethoxy-silane; Roth, Karlsruhe, Germany)-coated slides, air-dried for 30 sec, fixed in acetone at room temperature (22C, RT) for 1 min, air-dried for 15 sec at RT, and subjected to the staining procedure. No attempt was made to block endogenous peroxidase.
Primary Antibodies
The primary antibodies used are listed in Table 1. For positive controls and the determination of the optimal dilution of the primary antibodies, tissue known to contain the antigen in question was used. For negative controls the primary antibody was replaced with mouse or rabbit IgG at the appropriate dilution.
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Routine IHC Procedures
For the routine IHC procedures, frozen sections were stained by the HRP or LSAB method. The tissue sections (prepared as described above) were rehydrated in Tris-buffered saline (TBS; 25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl) for 5 min at RT. They were then incubated with the primary antibody in "antibody diluent" (DAKO) for 30 min at RT, rinsed three times in TBS, and subjected to the detection reaction. For the HRP method, the sections were incubated with the HRP-labeled goat anti-mouse antibody or goat anti-rabbit antibody (both DAKO; dilution 1:100) for 30 min at RT. The sections were then washed three times in TBS and incubated at RT with 3,3'-diaminobenzidine (DAB) (Sigma Fast DAB tablet dissolved in deionized water; Sigma, Deisenhofen, Germany) for 5 min or 3-amino-9-ethylcarbazol (AEC-substrate solution, ready to use; DAKO) for 10 min. For the LSAB method, the sections were incubated with the biotinylated secondary antibody and HRP-conjugated streptavidin (both "ready to use"; DAKO) for 10 min, being gently rinsed in TBS between the incubation steps. The same chromogens were used as for the HRP method.
Rapid EnVision IHC Procedure
For details of the procedure, see Fig 1. The sections were incubated with the primary antibody in "antibody diluent" (DAKO) and goat-anti-mouse or goat anti-rabbit EnVisionHRPenzyme conjugate was performed for 3 min each. The "highly sensitive 3,3'diaminobenzidine plus" (DAB+) and the "3-amino-9-ethylcarbazol plus" (AEC+) chromogens (both from DAKO) were used as substrates for the EnVisionHRPenzymes. Staining intensity was further enhanced by modifying the manufacturer's protocol in that all incubation steps (primary antibodies, EnVision, and substrate reactions) were performed on slides placed horizontally on a thermal plate at 37C. After each incubation, the slides were dipped in TBS or, after the substrate reaction, in tapwater at RT and waved at maximum speed for 10 sec. Excess liquid (buffer/water) was soaked up by a paper towel. After the last wash in tapwater, the slides were dipped quickly in distilled water before counterstaining with Meyer's hematoxylin (Sigma; 15 sec), followed by 30 sec in hand-hot (42C) tapwater. The sections were mounted in Aqua Tex (Merck; Darmstadt, Germany) and examined with a light microscope (Leica). Staining was evaluated by three independent observers (AMG, PR, and TR) and graded from 0 to 3+ according to its intensity and specificity, taking into account the results obtained with the standard IHC procedures (HRP, LSAB).
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Results |
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The results are summarized in Table 1. The rapid EnVision procedure produced strong and specific staining that was clearly distinguishable from the weak background staining with most of the primary antibodies applied. To obtain optimal staining with the rapid EnVision protocol the primary antibodies had to be applied at concentrations four- to tenfold higher than in the standard IHC procedures. When the rapid protocol was performed with the same concentrations as in the standard procedures, staining was at best very weak and was often absent. Tissue morphology after staining with the modified protocol was almost identical to that observed by the HRP and LSAB methods, and no conspicuous freezing artifacts were noted.
Carrying out the incubations with the primary and secondary antibodies and both chromogens on a standard heating plate at 37C resulted in a marked increase in staining intensity, thus improving the sensitivity of the procedure. The higher temperature of the hand-hot tapwater accelerated the "blueing reaction" of the Meyer's hematoxylin in comparison with the use of cold water. Washing with TBS after the substrate reaction produced slightly more background than when tapwater was used, so we modified the EnVision manufacturer's protocol at this point also.
Because of the extremely short incubation times, humid chambers were not required to avoid evaporation of the immunoreagents. No nonspecific staining was observed in the control sections, except for staining of mast cells that were immunoreactive with a wide variety of different antibodies, a phenomenon that has been attributed to nonspecific binding of primary antibodies through ionic linkage (
Specific, evaluable, and reproducible immunostaining (graded from 1+ to 3+; see Table 1) was seen with 38 of the 45 antibodies applied using the rapid EnVision protocol (Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7). When the results were inconsistent, the mean value of the three individual results was taken. With some antibodies (against CD14, CD45, CD56, cathepsin D, epithelial membrane antigen, and smooth muscle actin), EnVision produced even less background staining and a better signal-to-noise ratio than the routine HRP or LSAB procedures. As a substitute for the two clones for estrogen and progesterone receptors and the monoclonal anti-CD3 and c-erbB2 antibodies, which failed to stain with the rapid protocol, other antibodies were available that produced useful staining results. The polyclonal antibody against CD3 needed an extended incubation time of 5 min to produce sufficient staining. The two polyclonal antibodies against S100 protein did not produce specific staining on cryostat sections, either with the rapid EnVision protocol or with the standard IHC procedures.
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Discussion |
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Because in some cases it would be useful to be able to perform rapid immunohistochemical investigations, in addition to staining with hematoxylin and eosin, during intraoperative frozen section diagnosis, several studies have tried to reduce the time for immunostaining using various different techniques (
In this study, we found that a broad spectrum of antigens could be detected in frozen sections within less than 13 min, using a modified protocol for the EnVision system. Therefore, this rapid, simple, and sensitive immunostaining method can be used to greatly enhance the diagnostic information obtained by standard frozen section examination during surgery. A limitation of the procedure is the number of slides that can be handled. However, in our experience, two slides with two sections each, allowing the simultaneous detection of up to four different markers, produce sufficient information for intraoperative histopathological diagnosis in most cases. The staining procedure presented here is simple and timesaving, requires no special equipment, and can easily be adapted to different applications in routine diagnostic work and research. There is no need for the microwave treatment used in some other rapid IHC protocols that have been described (
For most antibodies, staining results with the rapid modified EnVision were comparable to those obtained using the standard and time-consuming LSAB method (
In summary, the rapid EnVision protocol presented here allows immunostaining of frozen sections in less than 13 min. This method could represent a useful new tool in surgical pathology and research, enabling more accurate frozen section diagnosis than staining with hematoxylin and eosin alone.
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Acknowledgments |
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Supported by a grant from the Federal Ministry of Education and Research (01KS9603) and by the Interdisciplinary Center of Clinical Research Würzburg (IZKF).
This work is dedicated to Professor Edwin Kaiserling on the occasion of his 60th birthday.
Received for publication January 12, 2001; accepted January 17, 2001.
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