Application of Cryotechniques with Freeze-substitution for the Immunohistochemical Demonstration of Intranuclear pCREB and Chromosome Territory

Abstract

Intranuclear localization of signal molecules and chromosome territories has become more attractive in relation to postgenomic analyses of cellular functions. Cryotechniques and freeze-substitution (CrT-FS) have been generally used for electron microscopic observation to obtain better ultrastructure and immunoreactivity. To investigate benefits of applying the CrT-FS method to immunostaining of intranuclear signal molecules and FISH for chromosome territories, we performed an immunohistochemical study of phosphorylated cAMP-responsive element binding protein (pCREB) in mouse cerebellar tissues and a FISH study of chromosome 18 territory in human thyroid tissues using various cryotechniques. The immunoreactivity of pCREB was more clearly detected without antigen retrieval treatment on sections prepared by the CrT-FS method than those prepared by the conventional dehydration method. In the FISH study, more definite probe labeling of the chromosome territory could be obtained on paraffin sections by the CrT-FS method without microwave treatment, although such labeling was not clear even with microwave treatment on sections prepared by the routine dehydration method. The CrT-FS preserved relatively native morphology by preventing shrinkage of nuclei, and produced better immunoreactivity. Because the reduction of routine pretreatments in the present study might reveal more native morphology, the CrT-FS method would be a useful technique for intranuclear immunostaining and FISH.

Keywords

quick freezing freeze-substitution in vivo cryotechnique antigen retrieval pCREB FISH chromosome territory

T he cryotechnique and freeze-substitution (CrT-FS) method has been commonly used for electron microscopy to minimize ultrastructural and immunoreactive changes of cells and tissues (Menco 1986). This is because ultimately prompt immobilization of molecules and structures, which is known to be most characteristic of the cryotechniques (CrT), can be hardly achieved by any kinds of routine chemical fixation steps. Recently, our “in vivo cryotechnique” followed by the freeze-substitution (FS) has been developed to overcome problematic ultrastructural changes of cells and tissues caused by cessation of blood circulation and resection of animal organs (Ohno et al. 1996,2001; Terada et al. 1998; Xue et al. 1998; Yu et al. 1998; Takayama et al. 1999,2000; Watanabe et al. 2000; Zea-Aragon et al. 2004a,b). Therefore, some ultrastructural and immunoreactive problems might be improved to a considerable degree at the electron microscopic level by using the in vivo cryotechnique. However, the CrT-FS method has not as often been applied to immunohistochemical (IHC) studies by light microscopy as to those by EM (Shiurba 2001).

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.

Materials and Methods

Tissue Preparation for Immunostaining of pCREB in Mouse Cerebellum

Conventional Fixation and Alcohol Dehydration. The present animal experiment was approved by the University of Yamanashi Animal Care and Use Committee. Adult C57BL/6 mice were anesthetized with an IP injection of Nembutal and perfused with 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4. The cerebellum was removed, cut into small pieces, and immersed in the same fixative for 1 hr The fixed specimens were dehydrated in a graded series of ethanol and xylene and finally embedded in paraffin.

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 avidin-biotin-HRP solution (Vector Laboratories) for 1 hr and visualized with cobalt-enhanced diamino-benzidine 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 Al-exa594-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 hematoxylin-eosin (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 QF-FS 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 × 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 on-off 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.

Results

Immunostaining of pCREB in Mouse Cerebellum

The immunoreactivity of pCREB was not as clearly detected on paraffin sections prepared by conventional fixation-dehydration without microwave treatment (Figures 1a and 1f), but it was heterogeneously enhanced by antigen retrieval treatment with microwave irradiation (Figures 1b and 1i). Such immunore-activity was mainly detected in nuclei of most granule cells (Figure 1b, large arrows) and in some small neurons in the molecular layer (Figure 1b, small arrows). Some nuclei of Purkinje cells were also faintly immunostained for pCREB (Figure 1b, large arrowheads). On paraffin sections prepared by the other three cryotechniques, however, the immunoreactivity of pCREB was much more clearly detected without microwave treatment for antigen retrieval (Figures 2 and 3). The IHC distribution of pCREB on the sections prepared by the cryotechniques appeared to be similar to that of the conventional paraffin sections with microwave treatment, but nuclei in the sections prepared by cryotechniques are less shrunken than those in the sections prepared by the conventional method. Although the immunoreactivity of pCREB was slightly enhanced on some sections prepared by the QF of fresh tissues with microwave irradiation (Figures 2c versus 2d), such immunoreactivity was almost at a similar level (Figures 2a, 2b, 3a, and 3b). The immunoreactivity was also observed on paraffin sections prepared by the QF of fresh tissues (Figure 2e) and the in vivo cryotechnique of living cerebellar tissues (Figures 3c and 3d) without microwave treatment, even when the primary antibody against pCREB was used at a higher dilution of 1:1000.

Figure 1

Immunohistochemical staining of pCREB (a–d,f,g,i,j) and DAPI (e,g,h j) with (b,d,h–j) or without (a,c,e–g) microwave irradiation in serial sections of paraffin-embedded tissues prepared by conventional fixation-dehydration. The anti-pCREB antibody was used at a dilution of 1:250. Immunoreactivity of pCREB is mainly detected in nuclei of most granule cells (b, large arrows) and some neurons in the molecular layer (b, small arrows). Some nuclei of Purkinje cells are also faintly immunostained (b, large arrowheads). Note that the unclear immunoreactivity (a,f) is heterogenously enhanced by the microwave treatment (b,i). Immunofluorescent staining of pCREB cannot be detected in the areas stained brightly with DAPI, which appear to be nucleoli (i,j, arrowheads). No immunoreactivity can be seen in immunocontrols (c,d). ML, molecular layer; GL, granular layer; Mw, microwave treatment. Bars: a = 50 μm; e = 10 μm; large inset = 500 μm; small inset = 10 μm.

Figure 2

IHC staining of pCREB (a–d,e,g,h,j,k) and DAPI (f,h,i,k) with (b,d,i–k) or without (a,c,e–h) microwave irradiation in serial sections of paraffin-embedded tissues prepared by perfusion-fixation followed by quick-freezing (PF–QF) (a,b) or quick-freezing of fresh resected tissues (FQF) (c-k). The primary antibody was used at dilutions of 1:250 (a–d,g,h,j,k) or 1:1000 (e, designated as ×1000). Immunoreactivity obtained by PF-QF (a,b) and FQF (c,d) is detected more clearly than by conventional fixation-dehydration (Figure 1a) without microwave pre-treatment (Figures 1a and 1c). Immunofluorescent staining of pCREB could also be observed without microwave treatment (g). Immunoreactivity is still clearly detected at a higher dilution (X1000) of the primary antibody in FQF (e). Although the immunoreactivity appears to be slightly enhanced by the microwave irradiation in FQF (c vs d), such an enhancement is not constantly obtained, as in PF-QF (a versus b). At higher magnification, the nuclei in the sections prepared by PF-QF or FQF (small insets in a-d) appear to be less shrunken than those prepared by conventional fixation-dehydration (Figures 1a and 1b). As in the sections prepared by the conventional method, immunofluorescent staining of pCREB cannot be detected in the areas stained brightly with DAPI, which appear to be nucleoli (f-k, arrowheads). ML, molecular layer; GL, granular layer. Bars: a = 50 μm; f = 10 μm; large inset = 500 μm; small inset = 10 μm.

FISH for Analysis of Chromosome Territory in Human Thyroid

The histological preservation of normal thyroid follicles revealed by the QF-FS method (Figure 4b) appeared to be better than that by the conventional fixation-dehydration (Figure 4a), as the tiny free spaces were less remarkable in the sections prepared by the quick-freezing method. However, on the same section prepared by the QF-FS method, the morphology of thyroid tissues was not preserved as well in the badly frozen areas with ice crystals (Figure 4c). The intranuclear distribution of the chromosome 18 territory could not be clearly observed on paraffin sections prepared by conventional fixation-dehydration, even though microwave irradiation was performed to increase the permeability of the probes (Figure 4d). On the contrary, it could be always visualized clearly without microwave treatment on paraffin sections prepared by QF-FS (Figure 4e).

Figure 3

Immunostaining of pCREB with (b,d) or without (a,c) microwave irradiation in serial sections of paraffin-embedded tissues prepared by the in vivo cryotechnique. The primary antibody was used at dilutions of 1:250 (a,b ×250) or 1:1000 (c,d ×1000). Immunoreactivity of pCREB is also more obviously detected in the sections prepared by the in vivo cryotechnique (a,b) compared with conventional fixation-dehydration (Figure 1), even without microwave treatment (a). At higher magnification, the nuclei (small insets in a,b) are less shrunken than those prepared by conventional fixation-dehydration (Figures 1a and 1b). Immunoreactivity is also detected at a higher dilution (1:1000) of the primary antibody (c,d). ML, molecular layer; GL, granular layer. Bars = 50 μm; insets = 500 μm.

Figure 4

Light micrographs of sections with HE staining (a–c) and FISH analyses of chromosome 18 territory (d,e) in human thyroid tissues prepared by conventional fixation-dehydration (a,d) and QF–FS (b–e). In HE-stained sections, the morphological preservation by QF–FS (b) in an area without any light microscopically detectable ice crystals is better than that by the conventional method (a). But in another area with insufficient quality of quick-freezing (c), morphology such as that of thyroid follicles is destroyed by many ice crystals (c, arrowheads). With FISH analyses of the chromosome territory, the fluorescence reactivity is hardly seen even with microwave treatment in conventionally prepared paraffin sections (d). Conversely, in sections prepared by QF–FS (e) the fluorescence signals of the chromosome territory are more clearly detected (e, arrows) and their follicular distribution appears to be better preserved, even though microwave irradiation was not performed. Co, colloid in lumen. Bar = 50 μm.

Discussion

The intracellular nucleus has been a troublesome compartment for immunostaining because of masking or steric hindrance by crosslinking of compact chromatin components induced by the molecular crosslinking function of common aldehyde fixatives (Brandtzaeg 1982; Shintaku and Said 1987). Although various antigen retrieval techniques, including microwave irradiation, had been tried to improve such problems, most of these trials were only partially successful, depending on some identified factors and presumably on some unidentified ones (Cattoretti et al. 1993; Podkletnova and Alho 1993; Shi et al. 1995,2001). Moreover, it has sometimes been difficult to obtain full and constant retrieval effects even under the same conditions with microwave treatment (Norton 1993), although a great effort has been made to fix such problems (Evers and Uylings 1994,1997; Shi et al. 2001). In the present study, however, we could constantly obtain enhanced immunoreactivity of pCREB by using cryotechniques for tissue preparations. We also obtained the same enhancement using the anti-CREB antibody available from Cell Signaling Technology (Beverly, MA) (data not shown). We have considered that this finding is due not only to the preservation of native immunoreactivity in situ but also to exposure of hindered antigenicity with freezing, because such good immunoreactivity of pCREB could be also obtained by the QF-FS method after conventional 2% PFA fixation. One possible mechanism of this antigenicity enhancement might be dramatic improvement of antibody permeability induced by tiny intracellular ice crystals, which are usually not visible at the light microscopic level but are large enough to expose some epitopes within compact intranuclear components. This idea might be supported by the findings that, in the sections prepared by cryotechniques, no remarkable ice crystals could be observed in areas ~300-400 μm deep from the frozen tissue surface at the LM level (data not shown), and that the antigenicity enhancement induced by the cryotechniques was not remarkably affected by microwave treatment. These findings might indicate that the enhancement effects with both cryotechniques and microwave treatment share a similar mechanism. One mechanism of microwave antigen retrieval was proposed to be loosening of the crosslinked protein networks that blocked antibody penetration (Menco 1986; Shi et al. 2001). Considering such mechanisms, the cryotechniques would be more effectively used in the case of some intranuclear antigens exhibiting little immunoreactivity with other conventional preparation methods.

Next, considering such mechanisms of antigenicity enhancement as described above, it is reasonable that, in our present FISH study, the QF-FS 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 QF-FS method, fewer pretreatments were needed for good fluorescence labeling with FISH. Moreover, the QF-FS method can also be used for human clinical specimens, providing better structural preservation in situ than that with conventional fixation-dehydration, although such improvement should be more precisely and quantitatively analyzed in future studies. Therefore, the QF-FS 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 ultra-structure 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.

Footnotes

Acknowledgements

We would like to thank Drs Takeshi Baba, Yasuhisa Fujii, and Zagreb Zea-Aragon, Department of Anatomy, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, for constructive comments on this work.

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