Application of Cryotechniques with Freeze-substitution for the Immunohistochemical Demonstration of Intranuclear pCREB and Chromosome Territory
Departments of Anatomy (NO,NT,SO) and Pathology (SM,RK), Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho, Yamanashi, Japan
Correspondence to: Shinichi Ohno, MD, PhD, Professor and Chairman, Dept. of Anatomy, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Tamaho, Yamanashi 409-3898, Japan. E-mail: sohno{at}yamanashi.ac.jp
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Summary |
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Key Words: quick freezing freeze-substitution in vivo cryotechnique antigen retrieval pCREB FISH chromosome territory
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Introduction |
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The purpose of the present study was to clarify some benefits of the application of the CrT-FS method to LM immunostaining of intranuclear antigens and fluorescence in situ hybridization (FISH) on paraffin-embedded sections. We checked the immunoreactivity of an intranuclear functional protein, phosphorylated cAMP-responsive element binding protein (pCREB), in C57BL/6 mouse cerebellum by various cryofixation methods, either with or without a routine microwave treatment for antigen retrieval. Then we examined by FISH a chromosome territory of surgically resected human thyroid tissues that were prepared by the CrT-FS method or another conventional fixation method. On the basis of our results, the CrT-FS method was found to be successfully applied to LM immunostaining of intranuclear antigens and FISH, and also had another benefit, i.e., better immunostaining and probe labeling could be obtained without complicated pretreatments of biological specimens.
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Materials and Methods |
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Perfusion-fixation Followed by Quick Freezing (QF)
After the perfusion, pieces of cerebellar tissues were quickly frozen by plunging into liquid isopentane-propane cryogen (193C), as previously described (Zea-Aragon et al. 2004b). The volume ratio of isopentane to liquidized propane was usually between 1:2 and 1:3 to achieve the lowest temperature (193C) during the QF method (Jehl et al. 1981
). Then the frozen specimens were freeze-substituted as described below.
QF of Fresh Resected Tissues
After anesthesia, cranial bones of the mice were carefully opened without heavy bleeding by a dental electronic drill. The cerebellum was speedily removed, cut into small pieces with razor blades, and quickly frozen as described above. The frozen specimens were routinely freeze-substituted as described below.
In Vivo Cryotechnique
Our in vivo cryotechnique was developed and performed as described previously (Ohno et al. 1996; Zea-Aragon et al. 2004a
). Briefly, after anesthesia and exposure, the cerebellum was cut with a cryoknife precooled in liquid nitrogen (196C) under an in vivo cryoapparatus (IV-11; Eiko Engineering, Hitachinaka, Ibaragi, Japan) and the liquid isopentane-propane cryogen (193C) was simultaneously poured over it. The cerebellum frozen in vivo was removed by a dental electric drill in liquid nitrogen and routinely freeze-substituted as described below.
Freeze-substitution (FS) for Paraffin Embedding
The FS solution was prepared as follows. Twenty percent paraformaldehyde in distilled water (DW) was added to pure acetone to a final concentration of 2% and the solution was completely dehydrated by incubating overnight at 4C with an adequate amount of Molecular Sieves 3A with indicator (Nacalai Tesque; Kyoto, Japan) whose size was 2 mm. The solution was cooled at about 80C in dry ice-acetone just before use. The frozen specimens were transferred into the chilled FS solution and freeze-substituted for 48 hr, during which frozen tissue specimens in the FS solution were continuously cooled in dry ice-acetone. After 48 hr, the temperature of the specimens in the FS solution was gradually elevated to room temperature by transferring the specimens in FS solution into a deep-freezer (25C), a common freezer (4C) and finally onto a room table in sequence, with a 2-hr interval each. Then the freeze-substituted specimens were washed in pure acetone for a few hours, incubated in 100% ethanol 5 times for 15 min to 2 hr, depending on each specimen size, infiltrated in xylene twice for 30 min each, and routinely embedded in paraffin.
Immunostaining of pCREB in Mouse Cerebellum
The paraffin-embedded specimens were cut at 5-µm thickness, mounted on glass slides, deparaffinized, and rehydrated in PBS. Some sections were completely dipped in 10 mM sodium citrate buffer, pH 6.0, and irradiated at 700 W for 1 min and 350 W for 9 min in a microwave apparatus, followed by cooling at RT for 20 min. All sections were incubated with 1% hydrogen peroxide in PBS and then with blocking solution (3% normal goat serum in PBS) for 30 min and 3 hr, respectively. Normal goat serum was obtained from Vector Laboratories (Burlingame, CA). Sections were then incubated with rabbit polyclonal anti-pCREB antibody (Upstate Technologies; Lake Placid, NY) at dilution concentrations of 1:250 and 1:1000 at 4C overnight, and with biotinylated goat anti-rabbit IgG antibody (Jackson Immuno Research; West Grove, PA) at RT for 1 hr. Immunocontrol sections were incubated with the blocking solution instead cubated in avidinbiotinHRP solution (Vector Laboratories) for 1 hr and visualized with cobalt-enhanced diaminobenzidine in hydrogen peroxide buffer solution (ImmunoPure; Pierce Chemical, Rockford, IL) for 5 min. The immunostained sections were postfixed with 0.04% osmium tetroxide in 0.1 M PB.
For the immunofluorescent staining, after incubation with anti-pCREB antibody the sections were incubated with Alexa594-conjugated goat anti-rabbit IgG antibody (Molecular Probes; Eugene, OR), washed in PBS, embedded in Vectashield with DAPI (Vector Laboratories), and observed with a light microscope (BX-61; Olympus, Tokyo, Japan).
Tissue Preparation for FISH of Human Thyroid Tissues
Conventional Fixation and Alcohol Dehydration
The present study was performed in accordance with the Guidelines for Clinical Experiment, University of Yamanashi. Surgically resected thyroid tissues were obtained from patients with thyroid tumors who gave their consent. They were immediately cut with razor blades, and some tissue pieces were immersed in buffered 20% formalin for a few hours. Then they were routinely dehydrated in a graded series of ethanol and embedded in paraffin as described above.
QF of Fresh Thyroid Tissues
Other pieces of thyroid tissues were quickly frozen, freeze-substituted, and embedded in paraffin as described above. Normal thyroid tissues were histopathologically checked on paraffin sections with common hematoxylineosin (HE) staining.
FISH Protocol for Chromosome Territory
A protocol without pepsin digestion had been tried but no signal labeling could be obtained. Therefore, the general FISH protocol with pepsin digestion was used for the thyroid tissue specimens prepared by the QFFS method (Pinkel et al. 1986). To the contrary, the modified FISH protocol with both pepsin digestion and microwave treatment, by which sensitive and specific signals could be clearly detected, was used for the formalin-fixed and alcohol-dehydrated specimens (Kitayama et al. 1999
,2000
). Briefly, both paraffin-embedded specimens were cut at 4-µm thickness, mounted on glass slides, deparaffinized, and rehydrated in DW. The formalin-fixed and alcohol-dehydrated sections were immersed in 10 mM citrate buffer (pH 6.0), treated with microwave irradiation at 500 W for 20 min, and cooled at RT for 1 hr. Then all the sections were incubated with pepsin (0.3% pepsin/0.01 N HCl) at 37C for 30 min, fixed with 4% PFA in PB at RT for 5 min, dehydrated in ethanol, and incubated with 0.1% NP-40 in 2 x SSC) at 37C for 30 min. After rinsing in PBS and dehydration in a graded series of ethanol, hybridization solution containing FITC-labeled probes against human chromosome18 (STAR* FISH; Cambio, Cambridge, UK) was added to the glass slides, and DNAs of both samples and probes were simultaneously denatured on the glass slides by heating at 85C for 5 min. For the formalin-fixed and alcohol-dehydrated sections, intermittent microwave irradiation by an onoff switch with intervals of 3 sec on and 2 sec off was performed at 43C for 1 hr. Then the ISH was performed in a humidified chamber at 37C overnight. After rinsing, the FISH signals were observed under an epi-illuminescent fluorescence microscope BX50 (Olympus) and the fluorescence images were acquired by a scientific-grade cooled CCD camera (Sensys; Photometrics, Tucson, AZ), connected to a personal computer.
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Results |
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Discussion |
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Next, considering such mechanisms of antigenicity enhancement as described above, it is reasonable that, in our present FISH study, the QFFS method could reduce the need for several pretreatment steps to improve probe permeability in conventional preparation methods. Although the FISH method has been applied to various kinds of paraffin-embedded tissues, it is quite difficult to obtain consistently good fluorescence labeling without several pretreatments, such as enzymatic digestion and microwave treatments (Kitayama et al. 2000; Watters and Bartlett 2002
). However, such pretreatments should be reduced to a lower level to avoid unnecessary tissue damage because they can easily cause pronounced changes of molecular structure in the intracellular nucleus (Solovei et al. 2002
). In the present study with the QFFS method, fewer pretreatments were needed for good fluorescence labeling with FISH. Moreover, the QFFS method can also be used for human clinical specimens, providing better structural preservation in situ than that with conventional fixationdehydration, although such improvement should be more precisely and quantitatively analyzed in future studies. Therefore, the QFFS method would be useful for FISH studies on surgically resected and paraffin-embedded human specimens, and would also establish new histopathological and diagnostic criteria with more native morphology in situ and fewer artifacts.
In the present study, our in vivo cryotechnique was applied to the IHC analysis of pCREB in the mouse cerebellum. It has been well known that both ultrastructure and molecular distribution could be easily changed by stopping the blood supply, probably due to anoxia and rapid loss of blood volume and pressure (Ohno et al. 1996,2001
; Terada et al. 1998
; Xue et al. 1998
; Yu et al. 1998
; Takayama et al. 2000
; Watanabe et al. 2000
). In addition, such loss of blood supply to organs, which was inevitable with conventional chemical fixation or the common quick-freezing of fresh specimens, is considered to affect the immunoreactivity of various functional molecules, because ischemia is one of the stimuli that induce diverse responses in many organs (Jaeschke and Lemasters 2003
; Valen 2003
). Considering precise analyses of dynamically changing functional proteins, including signal molecules, would be one of the major challenges in the postgenomic era. A method capable of immobilizing molecules and structures in vivo would have growing importance for minimizing morphological and IHC changes. Our in vivo cryotechnique can prevent such changes by immediately freezing all intracellular molecules, immunoreactivities, and structures. Moreover, it is also considered to enable more detailed analyses of transient and dynamic phenomena in living animal organs in vivo. In addition, in our present study, the in vivo cryotechnique has been shown to have another advantage: enhancing immunoreactivity of some intranuclear signal molecules. For these reasons, the in vivo cryotechnique would be useful especially for the analyses of intranuclear molecules dynamically changed by various stimuli, such as pCREB, although our results in the present study support only the theoretical advantages of the technique and more studies are essential to confirm such hypotheses.
In conclusion, the cryotechniques followed by the FS method could not only preserve good morphology and immunoreactivity but also could reduce the need for complicated pretreatments for IHC and FISH that have thus far been necessary. Because the reduction of such pretreatments might decrease biological artifacts during the preparation steps and could also reveal new morphological and histopathological findings, they would be more useful and alternative techniques for intranuclear analyses with IHC and FISH. Moreover, the in vivo cryotechnique, having the same benefits as the other CrT, will enable us to reveal the dynamic changes of intranuclear molecules by cryofixing the molecular changes immediately without any ischemic stress, which was usually inevitable with conventional cryotechniques.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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