Co-Localization of Multiple Antigens and Specific DNA

Production of Radiation Bone Marrow Chimeric Rats

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, 10 8 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-paraformaldehyde 16 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 sequence 14 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 × 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 2× standard sodium citrate (SSC) (1× SSC = 0.15 mol/L NaCl, 30 mmol/L Na₃citrate, pH7). After hybridization, excess probe was eliminated by washing the sections for 3 × 20 minutes in 2× SSC. Further washing was in 50% formamide/1× 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.

Combined in Situ Hybridization and Immunohistochemistry on Single Sections

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.

Embedding and Fixation

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.

Embedding in LR White and LR Gold resulted in excellent morphology and good in situ hybridization sensitivity and resolution but light microscopic immunohistochemistry results were poor. On Lowicryl-embedded tissue sections, neither in situ hybridization nor immunohistochemistry resulted in any valuable positive light microscopic signal. GMA embedding resulted in rather soft blocks not useful to obtain semithin serial sections. Resin mixtures resulting in harder GMA blocks became very hot during embedding, thus destroying the antigens. In addition, antigens seemed to be unstable in GMA because some antigens were detectable immediately after embedding but not after storing the blocks for 3 to 4 weeks.

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) ▶ .

During the course of our studies, several steps of the in situ hybridization protocol were optimized. Microwave pretreatment in 10 mmol/L citrate buffer, pH 6.0, for 15 minutes and proteinase K pretreatment at 10 μg/ml for 5 minutes were optimal, 20 whereas other concentrations or incubation times negatively influenced the results. Detection of hybridized digoxigenated DNA probes by CARD and sensitive carbocyanine dyes was far superior to conventional detection strategies such as alkaline phosphatase-labeled secondary antibodies and nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate, 4-toluidine salt substrates. CARD combined with Cy-dyes was more sensitive, faster, better reproducible, and showed better optical resolution.

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 Iba1 21 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.

Immunohistochemistry and Co-Localization of Multiple Antigens in Human Sural Nerve Biopsies

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.