Tissues and animal models
Experiments were performed on male C57BL/6J Bommince mice (A/S Bomholtgaard, Ry, Denmark) weighing 25–28 g, and on adult male Sprague–Dawley rats (200–250 g; B and K Universal, Stockholm, Sweden). All animals were kept under standard conditions on a 12-hour daylight cycle with free access to food and water. Unilateral sciatic nerve transection (axotomy) was made on n=10 mice as described earlier . Surgical procedures were performed under anesthesia with isoflurane. Operated animals were allowed to survive for 2 weeks after surgery. Dorsal root rhizotomy was done on n=5 mice, and animals were allowed to survive for 10 days. Sciatic nerve ligation was done in n=5 mice, which were sacrificed after 10 hours. Human ganglia were harvested from children with obstetric brachial plexus lesions, and undergoing reconstructive nerve surgery. Human spinal cord was harvested from a 48-year-old women died from stroke. The studies have been approved by the local Ethical Committee for animal experiments (Norra Stockholms djursförsöksetiska nämnd), and experiments on hDRGs were approved by a local Ethical Committee with written consent from the next of kin.
mRNA detection in tissues
In situ hybridization analysis of Scgn mRNA in mouse olfactory bulbs using riboprobes was performed as previously described . Briefly, adult brains were perfusion fixed followed by post-fixation in the same fixative overnight (4% paraformaldehyde, in 0.1M PB), cryoprotected (30% sucrose in 0.1M PB), embedded in Tissue-tek OCT compound (Miles Laboratories, Elkhart, IN) and sectioned at a thickness of 20 μm. The fragment of scgn cDNA used for riboprobe synthesis was amplified from an adult mouse olfactory bulb cDNA preparation by PCR using Pfu DNA polymerase (Promega). The primers used for scgn cDNA amplification were flanked at their 5’ ends with T7 and SP6 polymerase acceptors 5’-[CTGTAATACGACTCACTATAGGG] TCTCTAAGGAAGGCCGCATA -3’ (sense) and 5’- [GGGATTTAGGTGACACTATAGA] AGACACAGTGCCAGCTCAGA -3’ (antisense). Resulting amplicons were directly used as templates for in vitro transcription. Amplicon size was confirmed by loading PCR products onto 1% agarose gels. Digoxigenin-labeled antisense and sense riboprobes for mouse scgn were synthesized by in vitro transcription using SP6 and T7 RNA polymerases (Roche). After synthesis, probes were cleaned by using the RNeasy kit (Qiagen) and DNA digested with RNase-free DNase I (Qiagen). Alkaline phosphatase-conjugated anti-digoxigenin antibodies (Roche, 1:2,000) were used with their signal developed by 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium as substrate (BCIP/NBT; Roche).
In situ hybridization using oligoprobes was performed as described previously . Briefly, a mixture of two commercially acquired oligonucleotide probes (CyberGene, Stockholm, Sweden, or MWG Biotech, Ebersberg, Germany) were used: 1) GGACAGGCAGAGCCATCTAACAGGGGAG, 2)ACACAGGGGCTTTCAGTGAGACAGGGATAGAT complementary to nucleotide sequences of the human Scgn (accession number NM_006998.3). hDRGs were air-dried and incubated with a hybridization solution containing 0.5 ng of labeled probe/slide. The hybridization solution contained 50% deionized formamide (J.T. Baker Chemicals, Deventer, The Netherlands), 4×SSC (0.6 M sodium chloride, 0.06 M sodium citrate), 1× Denhardt’s solution (0.02% bovine serum albumin, 0.02% Ficoll (Pharmacia, Uppsala, Sweden), 0.02% polyvinylpyrrolidone), 1% N-lauroylsarcosine, 0.02 MNaPO4 (pH 7.0), 10% dextran sulfate (Pharmacia), 500 μg/ml denatured salmon testis DNA (Sigma, St. Louis, MO, USA) and 200 mM dithiothreitol (LKB, Stockholm, Sweden). Sixteen hours after incubation the tissues were rinsed in 1×SSC for 4 times (each time 15 minutes) at 56°C and allowed to cool to RT, washed in distilled water, and transferred rapidly through 60% and 95% ethanol. The 33 P-dATP-labeled sections were apposed to β-max autoradiography film (Amersham). The films were exposed for one and half months and developed with Kodak LX 24 and fixed with Kodak AL4. The slides were rinsed in distilled water and coverslipped with glycerol. The sections were then counterstained with cresyl violet, dehydrated in graded series of ethanol, and coverslipped with Entellan (Merck, Darmstadt, Germany). All sections were examined in a Nikon Microphot microscope equipped for bright- and dark-field microscopy. Photographs were taken with a Nikon Coolpix 5000 digital camera (Nikon, Tokyo, Japan).
mRNA detection by quantitative real-time PCR
Quantitative PCR (qPCR) reactions were performed with custom designed primers according to published protocols . RNA isolated from tissues microdissected from adult C57Bl/6N mice (n = 3/DRG/spinal cord/olfactory bulb) were subjected to Scgn expression analysis after validating RNA integrity (data not shown). Gapdh was used to normalize Scgn mRNA expression levels. Results from qPCR experiments were subsequently compared to Scgn mRNA distribution as determined by in situ hybridization (primers: 5’CCCAGAAGTGGATGGATTTG 3’; reverse: 5’GTTGGGGATCAGGGGTTTAT 3’.
All operated (n=20) and control mice (n=10), as well as rats (n=10) were deeply anesthetized with sodium pentobarbital (10 mg/kg for mouse and 50 mg/kg for rat, both i.p.) and transcardially perfused with 20 ml (50 ml) of warm saline (0.9%; 37°C), followed by 20 ml (50 ml) of a warm mixture of 4% paraformaldehyde (37°C) and 0.4% picric acid in 0.16 M phosphate buffer (pH 7.2), and then by 50 ml (250 ml) of the same, but ice-cold fixative [79, 80]. The L 5 mDRGs, mTGs, rDRGs and rTGs, as well as the L4 and L5 segments of both mouse and rat spinal cord were dissected out and postfixed in the same fixative for 90 min at 4°C. Specimens were subsequently stored in 10% sucrose in phosphate buffered saline (PBS, 0.1 M, pH 7.4) containing 0.01% sodium azide (Sigma, St. Louis, MO) and 0.02% bacitracin (Sigma) as preservatives at 4°C for 2 days. The hDRGs and spinal cord were immersion-fixed for four hours in ice-cold fixative and rinsed as mentioned above. Tissues were embedded with OCT compound (Tissue Tek), frozen and cut in a cryostat (Microm, Heidelberg, Germany) at 10 μm (mDRGs), 14 μm (mTGs, rDRGs and hDRGs) or 20 μm (mouse, rat and human spinal cords) thickness and mounted onto chrome-alum-gelatin-coated slides. Thaw-mounted sections were dried at room temperature (RT) for 30 min and rinsed with PBS for 15 min. Sections were incubated for 18 hours at 4°C in a humid chamber with rabbit anti-Scgn antiserum (1:1,000); [15, 16, 60] diluted in PBS containing 0.2% (w/v) bovine serum albumin and 0.03% Triton X-100 (Sigma). In addition a monospecific polyclonal rabbit anti-human Scgn antibody was used . Briefly, purified recombinant Scgn (540 μg) was emulsified in complete Freund's adjuvant and injected subcutaneously into a rabbit followed by two more injections of incomplete Freund's adjuvant. Serum collected one month after the third immunization contained high titer antibody activity against the recombinant protein when tested by ELISA .
Immunoreactivity was visualized using the tyramide signal amplification system (TSA Plus; NEN Life Science Products, Boston, MA). Briefly, the slides were rinsed with TNT buffer (0.1M Tris–HCl, pH 7.5; 0.15 M NaCl; 0.05% Tween 20) for 15 min at RT, blocked with TNB buffer (0.1M Tris–HCl; pH 7.5; 0.15M NaCl; 0.5% DuPont blocking reagent) for 30 min at RT followed by a 30-min incubation with horseradish peroxidase-labeled swine anti-rabbit antibody (1:200; Dako, Copenhagen, Denmark) diluted in TNB buffer. After a quick wash (15 min) in TNT buffer, all sections were exposed to biotinyl tyramide-fluorescein (1:100) diluted in amplification diluent for approximately 15 min, and finally washed in TNT buffer for 30 min at RT.
For double-staining experiments, immunohistochemistry was carried out with cocktails of primary antibodies: Scgn (1:1,000) plus CGRP (1:10,000) , PV (1:400), CB (1:400), CR (1:400), TRPV1(1:500) (St. Cruz Biotechnology, St. Cruz, CA), GRP (1:500) (ImmunoStar, Hudson, WI) or PKCgamma (1:1,000) (St. Cruz Biotechnology), respectively, following previous published protocols  (the three antibodies to CaBPs were from Swant, Bellinzona, Switzerland). For triple stainings, Scgn (1:1,000) plus PV (1:400) and CB (1:400) or Scgn (1:1,000) plus CB (1:400) and CR (1:400). In addition, a group of Scgn-labeled sections was incubated with the IB4 from Griffonia simplicifolia I (GSA I; IB4; 2.5 μg/ml; Vector Laboratories, Burlingame, CA)  followed by incubation with a goat anti-GSA I antiserum (1:4,000; Vector Laboratories) and rhodamine red X-conjugated donkey anti-goat antibody (1:200; Jackson ImmunoResearch, West Grove, PA). hDRG slides were only processed for Scgn and CGRP or IB4, respectively. Finally, all slides were coverslipped with glycerol/PBS (9:1) containing 2.5% DABCO (Sigma) [83, 84].
The specificity of Scgn antiserum was tested by preabsorption of the antiserum with homologous antigen at a concentration of 1 and/or 10 μM for 24 hours at 4°C. After incubation with control serum, i.e. Scgn antiserum pre-absorbed with the excess of Scgn, no fluorescent neuronal cells could be observed (data not shown).
Western blot analysis
L4 and L5 mDRGs from mice with unilateral sciatic nerve transection (n=5) and controls (n=5) were removed and immediately put on dry ice. DRGs and spinal cord (L4-5 segments) were homogenized in lysis buffer (50 mM Tris–HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM NaF and 1 mM Na3VO4) containing protease inhibitor cocktail (Sigma) using sonication. Lysates were centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected for western blots. Protein concentration was measured by Bradford’s Assay (Bio-Rad, Hercules, CA). Laemmeli sample buffer containing about 20 μg of protein was loaded in each lane and separated on 10% SDS-PAGE gel, then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Hemel, Hempstead, UK). The membranes were blocked with 5% non-fat dry milk in PBS with 0.1% Tween-20 for 1 hour at RT and incubated overnight at 4°C with an antibody against Scgn (for DRG sample, rabbit polyclonal, 1:1,000; for spinal cord sample, mouse monoclonal, 1:1,000; Atlas antibody clone 13B8). The membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at RT (1: 5,000-1: 10,000, DAKO) followed by ECL solution for 5 min (Amersham Biosciences, Piscataway, NJ) and exposed to X-ray film (NEN PerkinElmer, Waltham, MA). The membrane was stripped and re-probed for β-Actin (mouse monoclonal, 1: 5,000-10,000, Cell Signaling Technology, Danvers, MA) as loading control. Images were quantified with Quantity One software on non-saturated images (Bio-Rad).
Image analysis and quantification
Specimens were analyzed on a Bio-Rad Radiance Plus confocal scanning microscope (Bio-Rad, Hemel, Hempstead, UK) installed on a Nikon Eclipse E 600 fluorescence microscope (Nikon, Tokyo, Japan) equipped with x10 (0.5 numerical aperture, NA), x20 (0.75 NA) and x60 oil (1.40 NA) objectives. Fluorescein labeling was excited using the 488-nm line of the argon ion laser and detected after passing a HQ 530/60 (Bio-Rad) emission filter. For the detection of lissamine rhodamine sulfonyl chloride and rhodamine, the 543-nm HeNe laser was used in combination with the HQ 570 (Bio-Rad) emission filter. For the detection of DAPI a 405-nm laser was used. All the slides were scanned in a series of 1μm-thick optical sections. Consequently, images were analyzed separately and merged to evaluate possible colocalization. In some cases a Zeiss laser scanning microscope 780 system with a plan-apochromat x 20 (0.8NA) M27 objective was used.
To determine the percentage of IR NPs, the counting was performed on 10 or 14 μm thick sections, and every 4th or 6th section was selected (Nike Microphot-FX microscope, 20x objectives). The total number of immuno-positive NPs was divided by the total number of propidium iodide-stained  NPs in the DRG sections, and the percentage of positive NPs was calculated. Five to ten sections of each DRG from five animals in each group were included in the analysis, and 1,200-3,000 NPs were counted in each ganglion. The size distribution of NPs with a visible nucleus was measured using the Nikon Eclipse E 600 fluorescence microscope with Wasabi Image Software. We divided the NPs into small (a somal area of 100–600 μm2); medium-sized (600–1400 μm2) and large (>1400 μm2) according to earlier studies [76, 86]. The percentages of DRG NPs in each of these categories were calculated.
Differences between the percentage of Scgn-IR NPs as well as the gray levels of Scgn in mDRG neurons in ipsilateral and contralateral samples were evaluated by Student’s t test.