TECHNICAL NOTE |
Correspondence to: Denise A. Bessert, Dept. of Anatomy and Cell Biology, Wayne State Univ. School of Medicine, 540 E. Canfield St., Detroit, MI 48201.
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Summary |
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We applied in situ hybridization and the TUNEL technique to free-floating (vibratomed) sections of embryonic and postnatal mouse CNS. Full-length cDNAs specific for oligodendrocyte- or astrocyte-specific genes were labeled with digoxigenin using the random primer method. With paraformaldehyde-fixed sections, the nonradioactive in situ hybridization method provides detection of individual, very small glial progenitor cells in embryonic development. Small, isolated cells expressing oligodendrocyte specific messages can be detected in the neuroepithelium at embryonic and postnatal stages. The technique can be completed within 3 days and is as sensitive as the radioactive method. Likewise, the TUNEL method using DAB as the chromogen on free-floating sections provides excellent resolution. These DAB-stained sections can be embedded in plastic and thin-sectioned to visualize the ultrastructure of apoptotic cells. Both in situ hybridization and TUNEL methods can be applied to the same section, the tissue embedded in plastic, and semithin sections cut. The high resolution obtained with this combined procedure makes it possible to determine whether brain cells expressing glia-specific messages are undergoing apoptosis. (J Histochem Cytochem 47:693701, 1999)
Key Words: neuroglia, oligodendrocytes, myelin basic protein, proteolipid protein, glial fibrillary acidic protein, TUNEL, in situ hybridization, vibratome sections
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Introduction |
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Astrocytes and oligodendrocytes comprise the macroglia of the central nervous sytstem (CNS). These cells are considerably smaller than neurons, being on the order of 1020 µm (
We have found that the use of the nonradioactive in situ hybridization technique with free-floating (vibratomed) sections is extrememly sensitive and provides excellent cellular resolution of glia-specific mRNAs. These free-floating sections can be embedded in plastic and semithin-sectioned to provide even better resolution. Because of our interest in glial cell death during development (
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Materials and Methods |
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Tissue Preparation
Timed-pregnant C57BL/6J mice were obtained from Charles River Labs (Wilmington, MA). The first day of gestation was defined as the day the vaginal plug was found. Postnatal mice were obtained from our breeding colony maintained by the Division of Laboratory Animal Resources, a federally approved laboratory facility. Embryonic (E1420) and 074 days postnatal (DPN) mice were anesthetized with chloral hydrate and then perfused under pressure with fresh 4% paraformaldehyde (Electron Microscopy Sciences; Fort Washington, PA) in 0.1 M PBS, pH 7.4. Brains and cervical spinal cords were removed immediately after perfusion and placed in fresh 4% paraformaldehyde overnight at 4C. Fifty µm transverse sections were cut with a vibratome (Technical Products International; St Louis, MO) and kept in 4% paraformaldehyde in 24-well plates until used for in situ hybridization or TUNEL studies. Gloves were worn during sectioning for in situ hybridization, but after this step no additional sterile procedures were used.
In Situ Hybridization
Proteolipid protein (PLP) cDNA clone 68 (
Sections were washed in 0.1 M PBS, pH 7.4, three times for 10 min at room temperature (RT), treated with 0.3% Triton X-100 (ICN Biomedicals; Aurora, OH) in 0.1 M PBS for 15 min at RT, and washed in 0.1 M PBS. The sections were treated with proteinase K (GIBCO; Grand Island, NY) 0.3 µg/ml in 0.1 M Tris, pH 8.0, 0.05 M EDTA for 20 min at 37C. The sections were washed in 0.1 M glycine in 0.1 M PBS, pH 7.4, for 5 min at RT, washed in 0.1 M PBS for 1 min, and then incubated in 0.25% acetic anhydride (Sigma; St Louis, MO) in 0.1 M triethanolamine, pH 8.0, and 0.9% NaCl for 10 min at RT. After a wash in sterile dH2O for 10 min, the sections were incubated with prehybridization solution (50% formamide, 2 x SSC, 0.05 g/ml dextran sulfate, 1 x Denhardt's, and 0.1 mg/ml salmon testis DNA) for 1 hr at 52C. The prehybridization solution was then replaced with hybridization solution.
Sections were incubated overnight for 1216 hr at 52C. Sections were washed twice for 30 min at 37C in 50% formamide, 2 x SSC, then washed two more times in 50% formamide, 1 x SSC for 30 min at 37C. The final washes consisted of 1 x SSC for 30 min at RT and 0.5 x SSC for 30 min at RT. The sections were then treated with S1 nuclease (GIBCO) 700 U/100 µl solution for 15 min at 37C. Next, they were washed in 0.1 M Tris twice for 30 min, pH 7.5, incubated in anti-DIG (Boehringer Mannheim) at 1:1000 in 1 x Denhardt's in Tris, pH 7.5, for 1 hr and 15 min, then washed four times for 15 min in Tris, pH 7.5, at RT. The sections were next washed in Tris, pH 9.4, NaCl:MgCl2 (20:1) for 2 min, and color-developed in Tris, pH 9.4, NaCl:MgCl2 (20:1) in the presence of nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Boehringer Mannheim) for 30 min to 12 hr. Sections were rinsed several times and then mounted on glass slides with Aqua Poly/Mount (Polysciences; Warrington, PA).
Controls for in situ hybridization were the addition of hybridization solution without the DNA probe during the hybridization step. No staining was found in the control tissue. Our previous in situ hybridiztion studies (
TUNEL Procedure
Tissue used for the TUNEL procedure was treated in the same manner as described for in situ hybridization, up to the first dH2O step. After this step the tissue was washed in 0.1 M PBS, pH 7.4, for 5 min, treated with 0.2% H2O2 for 20 min, rinsed in 0.1 M PBS for 5 min, and then incubated in TUNEL reaction mixture using the In Situ Cell Death Detection Kit, POD (Boehringer Mannheim) for 1 hr at 37C. The tissue was then rinsed in 0.1 M PBS three times for 5 min and incubated in Converter-peroxidase (POD) from the In Situ Cell Death Detection Kit (Boehringer Mannheim) for 30 min at 37C, rinsed in 0.1 M PBS three times for 5 min, and color-developed with SIGMA FAST, a diaminobenzidine (DAB) POD substrate (Sigma). If TUNEL-treated sections were subsequently used for in situ hybridization, they were rinsed in 0.1 M PBS twice for 5 min, washed in dH2O for 10 min, and then incubated in prehybridization solution and the remaining steps performed as stated above.
The Boehringer Mannheim TUNEL kit was compared to the ApopTag TUNEL kit (ONCOR; Gaithersburg, MD). Tissue preparation was the same for both kits. For the ApopTag kit, the tissue was placed in the Equilibration Buffer (ONCOR) for 5 min, incubated in working-strength terminal deoxynucleotidyl transferase enzyme (TdT) for 1 hr at 37C, incubated in working-strength stop/wash buffer for 30 min at 37C, rinsed in 0.1 M PBS three times for 5 min, and then developed with SIGMA FAST, DABPOD substrate (Sigma). The tissue was then treated for in situ hybridization as described above. Both TUNEL kits yielded similiar results, a finding similar to a recent report comparing different TUNEL kits (
Controls for the TUNEL procedure were treated in the same manner as the test samples except that the TdT enzyme was omitted from the reaction mixtures in both kits and was replaced with dH2O. No labeling was found in the controls.
TUNEL-processed or combined TUNELin situ sections were further processed for plastic embedding in Araldite 502 (Electron Microscopy Sciences) plastic. Sections were rinsed several times in 0.1 M Tris, pH 7.5, dehydrated in a graded series of ETOH followed by three changes in 100% propylene oxide over 30 mins, and stored overnight in 50% propylene oxide/50% Araldite 502 in a dessicator at RT. The next day the Araldite was replaced with fresh 100% plastic, kept dessicated at RT for 34 hr, and then embedded in Araldite. Two-µm sections were cut on an AO/Reichert Ultracut E (Reichert-Jung; Vienna, Austria) microtome. Silvergold ultrathin sections were cut from blocks of TUNEL-processed tissue and photographed on a JEOL 1010 transmission electron microscope. All light micrographs were taken on a Leitz microscope using Kodak 160T tungsten film or TMAX100.
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Results |
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Perfusion with 4% paraformaldehyde and routine fixation methods provided excellent preservation of tissue for both the in situ hybridization and TUNEL studies. Low-magnification pictures of brain and spinal cord showed intense labeling for myelin protein genes in the mouse CNS using 50-µm free-floating sections (Figure 1A and Figure 1B). The distribution of PLP- and MBP-labeled cells in the brain and spinal cord essentially overlapped from the earliest time point studied (E14) to the latest time point (P74). Embryonic sections processed for myelin gene expression were generally developed twice as long as postnatal sections to expose the labeled cells. This suggests that the amount of message was several-fold less in younger than in older animals. Expression of GFAP mRNA was also examined to ascertain whether the free-floating section method was amenable to study the time of expression of other CNS genes. Intense signal for GFAP message was found in P0 mice, but the distribution of GFAP-labeled cells (Figure 1C) was different from that of PLP- and MBP-labeled cells. At this age, GFAP mRNA was expressed in many cells near the midline. These GFAP+ cells were frequently present in the neuroepithelium bordering white matter tracts and near the gray matter adjacent to the piaglial limitans. These astrocytes had more oval or rectangular perikarya than the oligodendrocytes, which were usually spherical.
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Examination of 50-µm sections at higher magnification showed that the cytoplasm was intensely stained for both astrocyte- and oligodendrocyte-specific mRNAs, but the nucleus was unstained (Figure 1G). The distribution of MBP and PLP messages in oligodendrocytes was different, with MBP extending into the processes, whereas PLP message was confined primarily to the perikaryon. High-magnification photographs of MBP in situ hybridization slides showed that this message extended into the distal processes where they expanded to form the myelin sheath (Figure 1G). Along the oligodendrocyte processes, MBP message tended to be aggregated into distinct clusters. In contrast, PLP very rarely extended into the finer processes.
The low background and the well-preserved tissue permitted identification of message-expressing cells in precise relationship to other neural structures. For example, individual MBP+ and PLP+ cells were occasionally found scattered in the neuroepithelium of young adult animals, especially at the dorsolateral edge of the lateral ventricles (Figure 1D). Single, isolated PLP+ or MBP+ cells were found throughout the cerebrum in embryonic development (not shown). At E14E18, some of these oligodendrocyte progenitor cells were located within the neuroepithelium (Figure 1F) and some were found in presumptive white matter tracts, but most were located just outside the neuroepithelium. Although these isolated cells had very little cytoplasm, many of them exhibited a ring of dense staining around the nucleus.
Another major advantage of the perfused, free-floating sections is the ability to resolve individual cells and to quantify them. For example, a cluster of MBP+ cells could be identified at the base of the third ventricle at E17 (Figure 1E) and, by focusing through the section, it was possible to quantify the number of stained cells. The boundaries of the neuroepithelium were visible in this section, and in this photograph the MBP+ cells were not within the neuroepithelium proper but were found immediately adjacent to it. These cells had already begun to develop short processes that contain small amounts of message.
TUNEL staining using free-floating sections also provides excellent cellular resolution of apoptotic cells. An advantage of using free-floating sections is that the TUNEL-stained sections can be combined with other techniques, including in situ hybridization, or they can be embedded in plastic for ultrastructural analyses. TUNEL staining of mouse cerebrum during the first postnatal week showed massive numbers of apoptotic cells in certain regions of the cerebrum, whereas other areas of the cerebrum had virtually no TUNEL+ cells (Figure 2A). Higher-magnification photomicrographs (Figure 2B) of apoptotic regions showed that most TUNEL+ cells had a signet ring morphology characteristic of apoptotic cells found in other tissues (e.g.,
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The free-floating sections can be processed first for TUNEL staining and then for in situ hybridization. After completion of the TUNEL procedure, the sections were visualized under an inverted microscope to confirm that the TUNEL staining was strongly positive. These sections were embedded in plastic and resectioned at 12 µm to visualize the location of reaction product. mRNA was visualized as a blue reaction product within the cytoplasm of cells, whereas the TUNEL staining appeared as a brown reaction product (Figure 1H). TUNEL staining was rarely localized in the large MBP+ and PLP+ cells, suggesting that the vast majority of apoptotic cells are not mature oligodendrocytes expressing large amounts of myelin messages. However, small TUNEL+ cells sometimes exhibited a blue reaction product (either MBP or PLP mRNA) around their nuclei (Figure 1H), strongly suggesting that apoptosis in the oligodendrocyte lineage was primarily restricted to immature oligodendrocytes.
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Discussion |
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The nonradioactive in situ hybridization protocol used for tissue sections is essentially similar to the nonradioactive in situ hybridizaton procedure used on our glial cell cultures (
Nonradioactive in situ hybridization applied to free-floating sections offers several advantages compared to the radioactive method. With radioactive in situ hybridization, message-bearing cells that are small and scattered throughout a tissue are likely to be mistaken for background or difficult to identify as positively labeled. Accordingly, the distribution of message-bearing cells within a particular tissue may be incomplete. This is particularly the case with neuroglial progenitor cells which are very small and often are not clustered throughout the embryonic brain. Isolated message-bearing cells in the neuroepithelium of adult animals are also likely to be overlooked.
In the embryonic brain, clusters of cells expressing putative oligodendrocyte-specific messages have been described along the ventral neuraxis in embryonic brain using radioactive in situ hybridization ( receptor (PDGF
R)-, 2',3'-cyclic-nucleotide 3' phosphodiesterase (CNPase)-, MBP- and PLP/DM20-expressing cells, it is virtually impossible to determine numbers of message-bearing cells in a particular location and, most importantly, whether such cells are present in the neuroepithelium where oligodendrocyte progenitors must arise (e.g.,
Comparison of PLP/DM20 gene expression of embryonic spinal cord at E14E18 using the radioactive and nonradioactive methods shows a similar pattern of labeled cells (
In this study, 12-KB cDNAs were labeled with digoxigenin using the random primer method, whereas riboprobes and partial cDNA clones were radiolabeled and used in most other studies examining glial gene expression (e.g.,
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Acknowledgments |
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Supported by the National Multiple Sclerosis Society.
We thank Ms C. Perry for performing the light microscopic TUNEL staining.
Received for publication October 6, 1998; accepted December 10, 1998.
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