Materials and Methods

Antibodies

Monoclonal antibodies included mouse anti-human-p75^NTR-antibodies (American Tissue Culture Collection, clone HB8737, Levi et al., 1994) used as hybridoma supernatants (1:5) and mouse anti-CD57/HNK-1-antibodies (Sigma C 6680, Saint Louis, Missouri, USA; 1:500). The anti-HNK-1-antibody recognizes a sulfoglucuronyl epitope linked to several glycolipids, glycoproteins, and proteoglycans that have been implicated in cell-cell interactions (Kruse et al., 1984; Chou et al., 1987; Quarles, 1997; Bartsch et al., 1993). HNK-1 is expressed in many species ranging from insects to humans albeit at different levels (O´Shannessy et al., 1985; Villar-Cheda et al., 2006). The used antibody is derived from the VC1.1 hybridoma generated by fusion of mouse myeloma cells with splenocytes from BALB/c mice immunized with cat cerebral cortex homogenates (Arimatsu et al., 1987; Naegele and Barnstable, 1991). Anti-p75^NTR-antibody (hybridoma ME20.4) was generated by immunization of BALB/c mice with the WM245 human melanoma cell line and its specificity for p75^NTR has been established (Ross et al., 1984, Scarpini et al., 1988). Anti-p75^NTR -antibodies inhibited binding of iodinated NGF to its receptor, immunoprecipitated a 75kDa entity following metabolic labelling (Ross et al., 1984) and stained Western blots of human neuroblastoma and neuroepithelioma cell lines (Baker et al., 1989).

Polyclonal antibodies used were goat-anti-rabbit-olfactory marker protein (OMP)-antibodies (Wako Chemicals 544-10001, Richmond, VA, 1:3,000) and rabbit anti-myelin basic protein (MBP)-antibodies (Chemicon AB980, Temecula, CA; 1:800).

OMP as a highly specific marker for mature olfactory neurons is a cytoplasmic protein (19kDa) whose expression is phylogenetically well conserved across vertebrate species (Buiakova et al., 1994) and not turned on until the olfactory neuron axons have reached their final target in the olfactory bulb (Graziadei et al., 1980). For generation of the antiserum, introduced by Keller and Margolis (1975), goats received multiple injections of rodent olfactory marker protein (Keller and Margolis, 1976). The MBP sequence is highly conserved from mammals to chicken.

The isoforms (17.2, 18.5, 20.2, 21.5) of adult human MBP are generated by

differential splicing of a single mRNA transcript (Boggs, 2006) and are expressed in a developmentally regulated manner. The 18.5kDa isoform is predominantly expressed by mature oligodendrocytes and Schwann cells. The immunogen for the used antibody was human brain MBP (McTigue et al., 1998). The antibody whose specificity has been confirmed in many studies is suitable for specific labelling of myelinating Schwann cells and oligodendrocytes.

Histological procedures

Tissue was prepared from young adult Beagle dogs (4 to 6 month-old) not suffering from diseases affecting the nervous and respiratory system as determined by clinical and histopathological examination. The animals were treated according to the legal and ethical requirements of the University of Veterinary Medicine Hannover and tissue was collected at necropsy (Department of Pathology, University for Veterinary Medicine Hannover). Heads were skinned, transected sagittally along the nasal septum and fixed in neutral buffered paraformaldehyde (10%) overnight. Frontal sections (0.5 cm thick) were cut and decalcified in ethylenediaminetetraacetic acid (EDTA, 10%) and paraformaldehyde (5%, pH 7.4) for 8-14 days with several changes of the solution. Maxillary nerve and sympathetic trunk were fixed and treated with the same decalcifying reagents for the sake of comparability. After decalcification, the tissue was rinsed in distilled water and embedded in paraffin for enzyme immunohistochemistry.

Immunohistochemistry

Paraffin sections (4 µm thick) were cut and mounted on SuperFrost^® Plus slides (Menzel-Gläser, Menzel GmbH & Co KG, Braunschweig, Germany). Sections were deparaffinized in a descending series of ethanol. Endogenous peroxidase was blocked with H2O2 (0.5% in methanol) for 30 minutes at room temperature (RT). For visualization of p75^NTR, sections were pretreated with sodium-citrate buffer (10mM, pH 6.0) in the microwave (800W, 20min.). The sections were treated with normal goat and horse (OMP staining) serum (1:5) diluted in phosphate-buffered saline (PBS, pH 7.1) for 20 minutes at RT to reduce non-specific binding. Incubation with

the primary antibodies diluted in PBS containing bovine serum albumin (BSA, 1%) was done overnight (4°C). After washing, biotinylated goat-anti-mouse-antibodies (BA-9200) for HNK-1 and p75^NTR, horse-anti-goat-antibodies (BA-9500) for OMP and goat-anti-rabbit-antibodies (BA-1000, all: Vector Labs, Burlingame, CA) for MBP were applied (1:200, 45 min., RT) followed by the avidin-biotin-peroxidase (ABC) complex system (30 min, RT; Vectastain^® Elite ABC Kit, PK6100, Vector Labs, Burlingame, CA). Color development was done using 3,3-diaminobenzidine-tetrahydrochloride (DAB, 0.05%) and H2O2 (0.03%) in PBS followed by counterstaining with hemalaun. After rinsing in distilled water and dehydration sections were coverslipped (Roti^®Histokit II, Carl Roth GmbH +Co, Karlsruhe, Germany). Specificity controls included the replacement of primary antibodies by ascites from non-immunized BALB/c mice (monoclonal antibodies), normal rabbit serum (polyclonal rabbit antibodies) and normal goat serum (polyclonal goat antibodies).

Cell culture

Olfactory bulb, both maxillary and sciatic nerve and cervicothoracic ganglion were prepared from dogs (Beagle, 4 to 6 month-old) not suffering from a nervous and respiratory system disease, as determined by clinical and histopathologic examination. Tissue was collected at necropsy under sterile conditions at the Department of Pathology (University for Veterinary Medicine Hannover). Isolation of OECs was essentially done as described previously (Krudewig et al., 2006). Briefly, enzymatic treatment was done using trypsin (0.25%, PAA, Marburg, Germany) in DME medium (Gibco Life Technol., Eggenstein, Germany) and cells were mechanically dissociated in the presence of DNase I (0.05%, Roche Diagnostics, Mannheim, Germany) using a fire-polished Pasteur pipette (Krudewig et al., 2006).

Cells were cultured on poly-L-lysine (0.5mg/ml) under standard conditions (5%CO2, 37°C) in DME medium supplemented with fetal calf serum (FCS, 10%, PAA Marburg, Germany) and pencillin/streptomycin (1%, PAA, Marburg, Germany). 96-well microtiter plates and 25cm² flasks (Nunclon, Nunc, Germany) were used for immunocytochemical analysis and expansion, respectively. OECs were expanded

using heregulin-1ß (HRG-1ß). After 7-10 days, cultures had reached confluency and were used for antibody-based purification.

For isolation of trigeminal Schwann cells, maxillary nerves were separated from surrounding connective tissue and cut into segments of about 0.5 cm length. The epineural sheath was removed and the nerve segments were cut into pieces (1mm³) using a scalpel. Enzymatic treatment was done with trypsin, collagenase and hyaluronidase (1% each) for 45min. at 37°C. Tissue dissociation and culture conditions were identical to those used for OECs.

Immunofluorescence

Immunostaining was done either with live OECs and Schwann cells or following fixation with paraformaldehyde (4%). For cell surface staining, viable cells were incubated with the primary and secondary antibodies diluted in complete medium (DME medium, 10%FCS) for 15min. at 37°C/5% CO2 (Wewetzer et al., 2005). For simultaneous visualization of a monoclonal and polyclonal antibody, mouse and rabbit-specific antibodies (Jackson ImmunoResearch Laboratories, Dianova, Hamburg, Germany) conjugated to Cy3 and Cy2, respectively, were used, whereas two monoclonal antibodies were detected by the combined application of IgM- (1:100, TRITC, Southern Biotechnology Associates, Biozol, Germany) and IgG1 -specific (1:100, Alexa 488, Invitrogen Molecular Probes, Germany) antibodies.

Cultures were fixed with paraformaldehyde (4%) and nuclei were counterstained using bisbenzimide (Krudewig et al., 2006).

For the detection of intracellular antigens, cells were fixed with paraformaldehyde and permeabilized using Triton-X100 (0.25%) diluted in PBS containing bovine serum albumin (BSA, fraction V, 1%, Sigma, Germany). Incubation of primary and secondary antibodies was done in this solution each for 1h at RT. Double immunostaining was done using the antibody conjugates described above, nuclei were stained using bisbenzimide. Cultures were rinsed in PBS and inspected under fluorescent excitation. Specificity controls included the omission of the primary antibodies and the application of antibodies directed against intracellular antigens to

live cells. Cultures were inspected using an inverted microscope (Olympus IX-70, Olympus Optical Co. GmbH, Hamburg, Germany).

Antibody-based purification of trigeminal Schwann cells and olfactory bulb-derived OECs

Purification of trigeminal Schwann cells and olfactory bulb-derived OECs was done using magnet-activated cell separation (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) essentially as described previously (Wewetzer et al., 2005; Krudewig et al., 2006). Incubation of dissociated cells with anti-p75^NTR-antibodies (hybridoma supernatant, 1:5) and goat-anti-mouse IgG MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany, 1:20) was done in ice-cold PEB (PBS w/o Ca²⁺ and Mg ²⁺, 2µM EDTA, 0.5% BSA, fraction V, Sigma, Munich, Germany) for 15min. each. The dissociated cells were washed in PEB, pelleted by centrifugation (1,000 rpm, 5min.), and passed over a PEB-equilibrated MiniMACS column (Miltenyi Biotec, Bergisch Gladbach, Germany). After elution, cells were pelleteted by centrifugation (1,000rpm, 5min.) and seeded in complete medium (DME medium, 10%FCS) onto 96-well microtiter plates (Nunclon, Nunc, Germany) for further analysis or on flasks for expansion in the presence of heregulin-1ß (HRG-1ß, Krudewig et al., 2006).

Illustrations

Immunohistochemical images were obtained with the Olympus BX-41 digital camera microscope (Olympus Optical Co. (Europe) GmbH, Hamburg) and stored as tagged image file format (tiff) files using analySIS® 3.2 software (Olympus Soft Imaging Solutions GmbH, Münster). Cell culture images were acquired with the inverted fluorescence microscope Olympus IX-70 (Olympus Optical Co. GmbH, Hamburg, Germany) and microphotographs were taken using the PM-30 photo system (Olympus) and a color reversal film (Ektachrome 400). Slides were scanned on the Minolta DiMAGE Scan Multi Pro scanner (Konika Minolta Photo Imaging Europe GmbH, Unterföhring). Adobe® Photoshop® 7.0 (Adobe Systems, Inc., San Jose, CA) was used to prepare the figures and insets, with adjustment of contrast, brightness and sharpness if necessary.