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Technical Advance |
From the Department of Neurology,*
Westfälische
Wilhelms-Universität, Münster, Germany; and the
Department of Pathology,
Dartmouth
Medical School, Lebanon, New Hampshire
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methacrylates are acrylic resins that were investigated in the 1950s and 1960s for their usefulness in electron microscopic studies. More recently, methyl methacrylate (MMA) was rediscovered as an embedding medium for immunolabeling studies particularly on bone marrow trephines because decalcifying is not necessary before MMA embedding.6 Furthermore, the feasibility to perform in situ hybridization of mRNA sequences was demonstrated.7 In addition, several other resins have been tested for their potential as embedding media for in situ hybridization and immunohistochemistry procedures, with varying results.8
Considerable improvements in the sensitivity of histochemical procedures were recently achieved by both microwave-stimulated antigen retrieval9 and the use of novel detection strategies. Catalyzed reporter deposition (CARD)10,11 involves the deposition of biotinylated tyramide after peroxidation of its precursor, tyramine. Thus, signal enhancement is possible at sites of localized peroxidase activity, eg, because of a peroxidase-coupled antibody. Additional gains in sensitivity may be reached using novel fluorescent carbocyanine dyes.
Radiation bone marrow chimeric rats are widely used to trace hematogenous, bone marrow-derived cells in different tissues and to differentiate them from resident cells. Within the nervous system, radiation bone marrow chimeric rats have been used to investigate the origin of microglia within the central nervous system and resident endoneurial macrophages of the peripheral nervous system.12,13 However, detection of the differentiating markers, such as major histocompatibility complex haplotypes or CD45 haplotypes, depends on previous cytokine stimulation which may alter the biological behavior of such cells. In TK-tsa transgenic Lewis rats, there is stable integration of 250 to 300 copies of a functionally silent DNA sequence into the genome14 that is accessible to histochemical detection by in situ hybridization.5 This transgene may thus be used as a differentiating cellular marker in radiation bone marrow chimeric rats and may be detected without previous manipulation of the animals by cytokines.
In an attempt to characterize radiation bone marrow chimeric rats carrying the TK-tsa transgene, we developed a novel and highly sensitive technique to co-localize specific DNA and multiple antigens at the same tissue level using MMA-embedded tissue. We also applied this technique to diagnostic human sural nerve biopsies and were able to co-localize several antigens using a postembedding immunohistochemical procedure on serial semithin sections.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type Lewis rats and Tk-tsa transgenic rats were obtained from Charles River (Sulzfeld, Germany) and BRL (Füllinsdorf, Switzerland), respectively. Bone marrow chimeric rats were created as described previously.12 In brief, recipient rats were lethally irradiated with 1000 rads. Subsequently, 108 bone marrow cells from donor rats were transplanted into irradiated recipients by injection into the tail vein. Chimeras were created either way, ie, wild-type bone marrow was transplanted into irradiated Tk-tsa rats and vice versa. Rats were allowed to recover for 3 months before sacrifice. Nerve crush experiments were performed as described previously.15 Some rats were injected intraperitoneally with 75 mg/kg of bromodeoxyuridine 2 hours before sacrifice.
Fixation and Tissue Preparation
Rats were deeply anaesthetized with medical ether and either decapitated or perfused through the left ventricle for 1 minute with a 6% hydroxyethyl-starch solution (HAES steril; Fresenius, Bad Homburg, Germany) followed by either 2% or 4% buffered paraformaldehyde at pH 7.4, 0.5 or 2% buffered glutaraldehyde at pH 7.4, or periodate-lysin-paraformaldehyde16 at pH 7.4 for 10 minutes. Tissue from perfused rats was postfixed in the same fixative for different time periods up to 24 hours.
Human sural nerve biopsies were obtained for diagnostic analysis after informed consent. Biopsies were fixed in 4% buffered paraformaldehyde at pH 7.4 for 24 hours. The embedding procedure described for rat tissue was performed without any modifications.
Cryostat and Paraffin Sections
For frozen sections, tissue was embedded in Tissue-tek (Sakura Finetek, Zoeterwoude, The Netherlands) and snap-frozen in isopentane cooled in liquid nitrogen. Cryosections (10-µm thick) were made on a Leica CM3050 cryotome (Leica, Bensheim, Germany) and mounted on coated slides. Paraffin embedding of fixed tissue was performed using standard methods. Five-µm thick sections were cut on a Leica SM2000R microtome and mounted on coated slides.
LR White, LR Gold, Lowicryl, and Glycol Methacrylate Embedding
Fixed tissue specimens were embedded in LR White, LR Gold, Lowicryl, and glycol methacrylate (GMA; all resins were purchased from Plano, Wetzlar, Germany) according to the manufacturer’s instructions. LR White blocks were either polymerized at 55°C for 3 days or, like Lowicryl blocks, under ultraviolet light for 24 hours. LR Gold was hardened at -20°C using an Osram 20 W halogen lamp at a distance of 10 cm for 24 hours. Sections were cut on an Reichert-Jung ultracut ultramicrotome. All sections were mounted on coated slides.
MMA Embedding
Rat tissue from brain, sciatic nerve, liver, thymus, and spleen, and sural nerve biopsy specimens were embedded as described,17 with some modifications. Tissue was washed in phosphate-buffered saline (PBS) for 15 minutes and then dehydrated through a graded ethanol series for 10 minutes each. Alternatively, tissue was dehydrated in pure acetone for 48 hours at -20°C. After dehydration, the tissue was placed in solution MMA1 consisting of 6 ml of MMA (Sigma, Deisenhofen, Germany), 3.5 ml of butyl-methacrylate (Sigma), 500 µl of methyl-benzoate (Merck, Darmstadt, Germany), and 120 µl of polyethylene glycol 400 (Plano) for 8 hours. The tissue was then incubated for 8 hours in solution MMA2 which is MMA1 containing an additional amount of 800 mg/100 ml dry benzoylperoxide (Plano). Polymerization was allowed for 48 hours at -20°C under vacuum in solution MMA3 containing the substances above plus 600 µl/100 ml N,N-dimethyl-p-toluidine (Merck). The blocks were cut on a Reichert-Jung ultracut ultramicrotome. Series of semithin sections were transferred onto coated glass slides and dried at 35°C for 2 hours. A series of tissue sections consisted of up to 16 sections.
Probes for in Situ Hybridization
A plasmid containing the Tk-tsa sequence14 was used as a template to prepare digoxigenin-labeled probes by polymerase chain reaction according to standard protocols. Five primer pairs were used to amplify different 200-bp regions of the transgene. The ratio of digoxigenin-11-dUTP to dTTP in the polymerase chain reaction mix was 1:5. Labeling efficiency was tested by dot blots of labeled polymerase chain reaction product compared with serial dilutions of a labeled standard sequence. All reagents needed for the digoxigenin-labeling and dot-blot procedure were purchased from Roche (Mannheim, Germany)
In Situ Hybridization
MMA was removed from the sections by incubation in 100% acetone for 3 x 20 minutes. Sections were washed in H2O and placed for 10 minutes in 0.25 mol/L HCl. They were then placed in 0.01 mol/L sodium citrate buffer, pH 6.0, and heated in a microwave oven at 850 W for 15 minutes Acetylation was performed for 3 minutes in 0.1 mol/L Tris-ethanolamine buffer at pH 8.0 with freshly added acetic anhydride at 0.5% final concentration. After washing in PBS and incubation in 1 mol/L NaSCN, protein digestion was achieved with 10 µg/ml proteinase K (Sigma) at 37°C for 5 minutes, followed by washes in H2O and PBS. No prehybridization step was included because preliminary experiments had not shown improvement of the results. For thermal denaturation, sections were heated at 95°C for 5 minutes. Hybridization was performed in a humid chamber at 37°C for 14 hours in a mixture containing the labeled DNA probe at 5 ng/µl, 5 mg/ml denatured salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 0.02% sodium dodecyl sulfate (all from Sigma) in 2x standard sodium citrate (SSC) (1x SSC = 0.15 mol/L NaCl, 30 mmol/L Na3citrate, pH7). After hybridization, excess probe was eliminated by washing the sections for 3 x 20 minutes in 2x SSC. Further washing was in 50% formamide/1x SSC at 50°C for 15 minutes.
The digoxigenated probe was detected by incubating the sections with a horseradish peroxidase-conjugated anti-digoxigenin antibody (DAKO, Hamburg, Germany) diluted 1:50 for 90 minutes, followed by incubation with 10 µg/ml biotinylated tyramide for 10 minutes in PBS containing 0.001% hydrogen peroxide and 0.1 mol/L imidazole (Sigma). Tyramine was biotinylated as described.18 The sections were incubated with fluorescent Cy3-streptavidine (1:100) for 30 minutes (Amersham, Braunschweig, Germany). Nuclear counterstaining was achieved using a fluorescence-saving mounting medium containing 4,6-diamidino-2-phenylindol (Vector Laboratories, Burlingame, CA).
Immunohistochemistry and Lectin Histochemistry
Sections were deplasticized as described above, placed in 10
mmol/L citrate buffer, pH 6, and heated in a microwave oven at 800 W
for 15 to 30 minutes depending on the respective antigen (see Table 2
).
Endogenous peroxidase and unspecific protein binding were sequentially
blocked with 3% H2O2 for
10 minutes and 10% bovine serum albumin for 20 minutes. Primary
antibodies and lectins, their dilutions and incubation times, all at
room temperature, are listed in Table 2
. After washing in PBS, a
secondary anti-mouse or anti-rabbit horseradish peroxidase-conjugated
antibody diluted 1:100 (DAKO) was used. All further steps were as
described above for in situ hybridization except that
fluorescent Cy2-streptavidin (Dianova, Hamburg, Germany) was used at
1:100 for 45 minutes instead of Cy3-streptavidin. Negative controls
included stainings without primary antibody or lectin, or with
irrelevant primary antibodies at comparable concentrations, and never
produced any staining apart from extremely low background.
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Sections were first treated as described for in situ hybridization. When the horseradish peroxidase-conjugated anti-digoxigenin antibody was added, the primary antibody for the immunohistochemistry procedure was added to the same mixture at the appropriate concentration. The procedure then followed the protocol above as described for the in situ hybridization procedure. After incubation with Cy3-streptavidine (Dianova), excessive biotin binding sites and horseradish peroxidase from the anti-DIG antibody were blocked by sequential 10-minute incubation steps with avidin, biotin (DAKO), and 3% H2O2. The further protocol was then as described for immunohistochemistry, beginning with the application of the horseradish peroxidase-conjugated secondary antibody. Negative controls included sections in which the primary antibody or the specific DNA probe were omitted.
Image Processing
Sections were examined with a Leica DM fluorescence microscope. Images were either digitized and transferred to a computer using a JVC 3 chip RGB video camera and a frame grabber PC extension card (Hasotec Technologies, Rostock, Germany), or a Diagnostic Instruments SPOT II camera system (Visitron, München, Germany). Merging of the detected fluorescent histochemical signals and the fluorescent nuclear staining was done by Adobe Photo Shop 4.0 or the SPOT II software. Assignment of signals on adjacent sections to specific cells was possible by comparing morphology and nuclear distribution patterns.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To obtain optimal results for immunohistochemistry, in
situ hybridization, and tissue morphology in co-localization
experiments we tested fresh-frozen sections, paraffin-embedded tissue,
and several resins in combination with different fixation regimes. The
results are summarized in Table 1
. In
paraffin-embedded and especially frozen material, combined
immunohistochemistry and in situ hybridization was feasible
on single sections but morphological impairment was considerable with
unsatisfactory assignment of the signals to individual cells.
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In contrast, MMA allowed for the combined use of immunohistochemistry and in situ hybridization with excellent morphological preservation. For immunohistochemistry applications, embedding in MMA could be further improved by modifying a previous protocol described by Erben.17 Dehydration of the tissue in pure acetone for 24 hours at -20°C, previously described as freeze substitution,19 lead to improved antigen preservation. Addition of methyl-benzoate to the methacrylate mix further improved immunohistochemistry sensitivity.
Among the fixation regimens tested, perfusion fixation with 4% buffered paraformaldehyde followed by immersion fixation for a further 3 hours for rat tissues and immersion fixation for 24 hours for human sural nerve biopsies provided an optimal compromise between morphology requirements and signal intensities.
In Situ Hybridization
Applying the in situ hybridization method described
above on MMA-embedded semithin sections, TK-tsa transgenic cell nuclei
were easily detected in TK-tsa transgenic Lewis rats (Figure 1)
. Tissue sections from wild-type Lewis
rats served as negative controls and never revealed labeled nuclei.
Nonspecific background signals were generally very low. On sections
from Tk-tsa transgenic rats, 
50% of the nuclei were labeled on a
single semithin section. Evaluation of three to four adjacent semithin
sections revealed additional signals on identified nuclei (Figure 1, A–D)
and increased the sensitivity to 85%. As the TK-tsa transgene
is integrated into the genome in multiple copies,14
some
nuclei showed multiple labels (Figure 1, A–D)
. In situ
hybridization performed on spleen tissue from bone marrow chimeric rats
produced by transplanting wild-type Lewis rat bone marrow into
irradiated TK-tsa transgenic Lewis rats or vice versa
revealed TK-tsa transgenic nuclei only in resident endothelial cells or
repopulating bone marrow-derived leukocytes, respectively (Figure 1, E and F)
.
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Immunohistochemistry
As our group focuses on neuroimmunology, we established
immunohistochemistry for several immune- and nervous system-related
antigens on MMA-embedded semithin sections of peripheral nerve, brain,
spleen, thymus, and liver (Figure 1, G–O)
. The antibodies, their
specificities, and the modalities of their use on MMA-embedded serial
sections are summarized in Table 2
. Using
the CARD technique in combination with microwave-stimulated antigen
retrieval and fluorescent Cy-dyes we could strongly enhance sensitivity
and signal resolution on MMA-embedded tissue. As in in situ
hybridization, conventional detection systems like
horseradish-peroxidase diaminobenzidine or alkaline-phosphatase fast
red detection were less sensitive and showed insufficient signal
resolution on MMA-embedded tissue.
Rat macrophages were successfully localized on MMA-embedded tissue
using antibodies ED1 (Figure 1G)
, KiM2R, and Iba121
and
the isolectin B4 of Griffonia simplicifolia (GSI B4, Figure 1H
). In contrast to paraffin-embedded tissue,22
pretreatment with neuraminidase was necessary to reveal the
lectin-binding sites. No positive signal could be obtained with
antibodies ED2 recognizing rat macrophage subsets and OX42 recognizing
rat complement receptor 3 on MMA-embedded tissue. On brain tissue, the
microglial and macrophage marker Iba1 revealed ramified microglial
cells (Figure 1I)
, and antibodies to glial fibrillary acidic protein
detected astrocytes (Figure 1J)
. Neurofilament protein was successfully
detected on peripheral nerve (Figure 1K)
by antibody NR4 against a
68-kd neurofilament. A monoclonal antibody against myelin basic protein
was used to detect myelin (Figure 1L)
. However, antigen retrieval was
required to detect normal uninterrupted myelin sheaths in peripheral
nerve. Without antigen retrieval, staining on MMA-embedded tissue
occurred mainly in disrupted myelin sheaths or myelin debris whereas
intact myelin sheaths showed only weak or no signal. Proliferation was
assessed using antibodies against proliferating cell nuclear antigen
and antibody MIB-5 against the Ki-67 antigen (not shown). In rats
pretreated with bromodeoxyuridine, proliferating cells were also easily
detected on MMA-embedded sections with the respective antibody
(Figure 1M)
. Finally, antibodies OX18 and OX6 detecting rat major
histocompatibility complex class I and II (Figure 1N)
, respectively,
were also found to be applicable on MMA.
Co-Localization of in Situ Hybridization and Immunohistochemistry Signals
Sequential application of tyramide amplification for in
situ hybridization and immunohistochemistry allowed detection of
in situ hybridization and immunohistochemistry signals on
the same tissue section (Figure 2, A–D)
.
Triple blocking of avidin, biotin, and horseradish peroxidase between
the tyramide amplification steps of in situ hybridization
and immunohistochemistry completely suppressed background signal and
nonspecific staining at sites of a previous histochemical reaction
(Figure 2, A–D)
. Stable antigens like bromodeoxyuridine, myelin basic
protein, or the ED1 antigen could easily be detected despite the
preceding in situ hybridization procedure. In contrast,
several less stable antigens could no longer be identified after
in situ hybridization. However, co-localization was easily
possible in such cases by performing in situ hybridization
and immunohistochemistry on adjacent 0.5-µm semithin serial sections
of MMA-embedded tissue (Figure 2, E–H)
. In this manner we could detect
in situ hybridization signal and up to three different
antigens at the same tissue level, and co-localization of even more
antigens should theoretically be feasible given the section thickness
of only 0.5 µm.
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The techniques developed on animal tissue were adapted for human
material without major changes (Figure 1K)
. Semithin sections of
MMA-embedded sural nerve biopsies stained with toluidine blue revealed
excellent morphological resolution far superior to paraffin sections.
However, contrast was not as good as on osmium-contrasted material
embedded in epon.
Immunohistochemistry with the respective antibodies revealed the localization of neurofilaments, myelin, major histocompatibility complex class II, T cells, macrophages, endothelial cells, and Schwann cells with excellent morphological resolution. As described for immunohistochemistry on rat tissue, conventional nonfluorescent detection systems were less sensitive than fluorescent detection systems and showed insufficient signal resolution on MMA-embedded tissue.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methacrylates have long been used as tissue-embedding media for electron microscopy23 and were recognized as useful embedding media for enzyme histochemistry, lectin histochemistry, immunohistochemistry, and nucleic acid hybridization already in the early days of these techniques.24 However, their popularity among researchers remained limited because of insufficient preservation of ultrastructural details and the greater ease of paraffin embedding for light microscopic studies in most tissues. Brain researchers in particular refrained from using methacrylates. In contrast, the methacrylate medium MMA is very useful as an embedding medium for bone marrow trephine biopsies as bony tissues do not need to be decalcified before embedding. More recently, MMA was rediscovered to be suitable for immunohistochemical analyses of bone and bone marrow trephine biopsies6,17,25-27 but also other tissues including lymphoid tissue26 and testis.28 Recently, a protocol for RNA detection by in situ hybridization was introduced, underlining the high preservation of cellular morphology of MMA-embedded tissue.7
In our experience, MMA proved to be the best of all embedding media tested both for histochemistry and morphological preservation. This good performance is because of several advantages, including ease of handling and tissue preservation. Another advantage is that it may be removed from tissue sections before the histochemical procedure. This "de-embedment" greatly improved the sensitivity of the immunohistochemical stains as previously described for in situ hybridization.7 Several modifications compared to previous protocols,17 including freeze substitution,29 further enhanced the sensitivity of our immunohistochemical procedures. However, as on paraffin sections, some antigens were lost after embedding in MMA in certain tissues. Whereas in bone marrow trephine biopsies, nearly all antibodies tested were reported to work in MMA-embedded tissue,6 we found several antibodies unsuitable for MMA-embedded sections. In our hands, antibodies suitable for paraffin sections also work reliably on MMA sections whereas those unsuitable for paraffin will not work on MMA either. In any case, fixation and antigen retrieval need to be reassessed for every antibody individually. Nevertheless, the reproducibility and superior quality of stains with those antibodies that do work make MMA very useful for immunohistochemical studies.
Another point of consideration are steps to improve signal intensity and resolution. Optimal results were obtained using CARD and fluorescent carbocyanine fluorophores as detection systems for both in situ hybridization and immunohistochemistry. CARD is a new signal amplification method mainly used to enhance the sensitivity of nonradioactive in situ hybridization protocols30,31 but was also used to detect antigens by immunohistochemistry on paraffin-embedded tissue.32 The carbocyanine dyes Cy2 (green) and Cy3 (red) proved to be more stable and exhibit stronger fluorescence than conventional fluorophores like fluorescein or rhodamine. In particular, the method was far superior to nonfluorescent detection methods based on color reactions visible with bright-field microscopy. In addition, background was extremely low using the methodology described, allowing for the use of relatively high antibody concentrations without any increase in background. Although lower concentrations comparable to those generally used on paraffin sections provided recognizable stains, higher antibody concentrations resulted in more brilliant stains without additional nonspecific binding.
In addition to its usefulness as an embedding medium for in situ hybridization and immunohistochemistry alone, MMA-embedded tissue sections were found to provide a good basis for double-labeling studies combining in situ hybridization and immunohistochemistry. This was possible on single sections despite using CARD detection twice with different fluorescent colors for in situ hybridization and immunohistochemistry, as all nonspecific binding could be blocked by means of sequential incubations with avidin, biotin, and hydrogen peroxide. After this blocking procedure, no additional nonspecific background was noted. Compared with our experience with conventional double-labeling studies,4 the results with MMA show better resolution with less background. Indirect tyramide amplification as used in the present study allows for a further increase in sensitivity and the detection of even smaller quantities of target compared with multitarget in situ hybridization based on direct tyramide amplification with fluorochrome-labeled tyramides as described in a previous report.11 Another advantage of MMA over paraffin or even frozen sections is the option to cut 0.5-µm semithin serial sections. Thus, multiple sections can be made through single individual tissue cells that can be stained for different antigens and nucleic acid targets without interference of the procedures. So far, we have co-localized transgenic DNA and three different antigens at the same tissue level on consecutive sections, but even more targets could be approached on further serial sections.
In contrast to experiments on MMA-embedded tissues, attempts to co-localize in situ hybridization and immunohistochemistry signals using frozen sections or standard embedding methods like paraffin were unsatisfactory. Problems included insufficient morphological preservation because of the necessary proteinase digestion steps, suboptimal signal-to-noise ratios, insufficient retainment of histochemical signals in double-staining experiments, and the technical limitations in cutting very thin frozen or paraffin sections. These problems were particularly prominent in peripheral nerve whereas other tissues showed better morphology and better retainment of signals. As paraffin and frozen sections yielded unsatisfactory results, the resins LR White, LR Gold, and Lowicryl, widely used for electron microscopy and immunoelectron microscopy,8,33 were also tested. With LR White and LR Gold, we achieved excellent morphology and well reproducible in situ hybridization signals, but immunohistochemistry signals were absent or extremely weak. This is in contrast to the usefulness of these resins for postembedding immunoelectron microscopy where gold-labeled antibodies are applied on ultrathin sections. In this situation, individual molecules of bound antibody can be detected, and access of the antibody to its antigen may be better. Embedding in Lowicryl resulted in good morphological preservation but only very low in situ hybridization signal intensity, probably because of crosslinking of DNA because of irradiation with ultraviolet light necessary to harden the Lowicryl blocks. Other researchers have successfully applied immunohistochemistry to epon-embedded sections34 or co-localized multiple antigens using serial semithin frozen sections cut at extremely low temperatures.35 However, epon sections allow immunohistochemistry only for very limited numbers of antibodies, and semithin cryostat sections are extremely difficult to handle and mechanically unstable.
Our method developed for co-localization of immunohistochemistry and in situ hybridization signals could be successfully adapted for the co-localization of multiple antigens in human sural nerve biopsies. Routine work-up for diagnostic sural nerve biopsies includes various stains on paraffin sections, toluidine blue-stained semithin sections of epon-embedded tissue for structural analysis, immunohistochemistry on cryostat, and paraffin sections to characterize inflammatory cells,36 and others. MMA embedding combines the availability of immunohistochemistry with excellent morphological resolution, which is far superior to paraffin and comes close but does not entirely reach the quality of epon-embedded semithin sections at the light microscopic level. The use of semithin serial sections again allows for the easy co-localization of multiple antigens at the same tissue level, which will be very useful for both diagnostic and research purposes in peripheral nerve disease. Although we have not tested in situ hybridization for specific DNA on human tissue yet, this should be possible as successfully as on rat tissue and may have many applications in tumor pathology and virology.
Our experiments were motivated by our search for finding an experimental bone marrow chimera system which would allow for tracing bone marrow-derived cells in the nervous system. The TK-tsa transgene contained in transgenic Lewis rats is an excellent cellular marker to identify and trace cells and to distinguish them from wild-type cells because it is not expressed and thus biologically inactive.14 Previously described cellular marker systems in radiation bone marrow chimeric rats are based on different MHC haplotypes which need stimulation by cytokines for sufficient expression levels to be detected immunohistochemically.12,13 As such treatment might alter gene expression and cellular reactivity, our new TK-tsa system has the great advantage that no pretreatment is necessary and no immunological alterations have to be feared. In addition, although the technique is demanding at first, cell tracing is very robust on MMA-embedded tissues once the histological settings are established. However, it should be noted that on sections as thin as 0.5 µm, the transgene may go undetected and may need additional serial sections through the nucleus for identification. The direction of bone marrow transplantation, ie, wild-type bone marrow into transgenic rats or vice versa, therefore will depend on the cell type under study. In our hands TK-tsa/wild-type bone marrow chimeric rats are extremely useful for studying cellular turnover and infiltration within the central and peripheral rat nervous system.
In conclusion, we describe a novel method to combine in situ hybridization and immunohistochemistry on single MMA-embedded tissue sections or on 0.5 µm semithin serial sections. This method allows for the detection of multiple DNA sequences and antigens at the same tissue level with high sensitivity and good morphological preservation. Given the versatility of the system, it should be useful wherever simultaneous detection of multiple antigens and nuclear acid sequences is required.
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Acknowledgements |
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Footnotes |
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Supported by the Deutsche Forschungsgemeinschaft (Ki532/3-1) and the IMF program, medical faculty, Westfälische Wilhelms-Universität Münster.
Accepted for publication August 31, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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