with 0.1% TritonX-100 (PBS-T) for 30 minutes at room temperature, and left in primary antibody diluted in PBS-T overnight at 4uC. Primary antibodies used for cell counting include those against TrkA (rabbit, 1:2000, gift of L.F. Reichardt), peripherin (rabbit, 1:1000, ab1530 Millipore and mouse, 1:1000, ab4573 abcam), and TRPV1 (guinea pig, 1:1000, GP14100 Neuromics), as well as the marker lectin IB4-FITC (1:200 in PBS containing Ca2+, L9381 Sigma). Type III Nrg1 was detected using a Type III-specific antibody generated in our lab (host = chicken) that recognizes an extracellular epitope of the protein (peptide sequence: ARTPEVRTPKSGTQPQTTET, corresponding to residues 195–214 of the Type III Nrg1 protein, KHL conjugated, Pocono Rabbit Farm & Laboratory Inc). Specific staining of motor neurons in the ventral horn of the spinal cord was used as a positive control (data not shown). This staining disappeared when the antibody was pre-incubated with the peptide against which it was generated, confirming the specificity of the staining in adult tissue (data not shown). Serum samples taken from the animal before it was immunized were used as a negative control and did not stain the spinal cord, dorsal root ganglia or hindpaw skin (data not shown). The following day secondary antibodies conjugated to Biotin (1:500, Jackson ImmunoResearch), Alexa488 (1:500, Molecular Probes), Alexa594 (1:500, Molecular Probes), Alexa546 (1:500, Molecular Probes) or AMCA (1:50, Jackson ImmunoR- esearch) (diluted in 10% NDS in PBS-T) were applied for 1 hour at room temperature. For immunofluorescence, sections were subsequently coverslipped using Vectashield (Vector Laboratories Inc, generally containing DAPI) and sealed with nail polish. For immunohistochemistry, sections were incubated with Avidin- Biotin Complex (ABC, Vector Laboratories Inc) and developed using a Diaminobenzidine (DAB) reaction (Vector Laboratories Inc), before being dehydrated and cleared with ethanol and xylene washes and mounted using Permount (Fisher Scientific). Co- localization of Type III Nrg1 with various sensory markers was assessed by acquiring confocal images of DRG sections using the Olympus Spinning Disk Confocal microscope (DSU; Olympus) equipped with a 406 oil Plan ApoN objective (1.42 NA), electron- multiplying charge-coupled device camera (Hamamatsu), and Slidebook software (Version 5; Olympus). To count DRG neuronal profiles immunoreactive for a particular sensory marker, epifluorescent images were acquired using an Axio Imager microscope (Carl Zeiss, Inc.) equipped with a 206 Plan-Apochromat objective (0.8 NA), a charge-coupled device camera (Hamamatsu) and Metamorph software (MDS Analytical Technologies). A constant exposure time was used for each sensory marker. Images were analyzed using Metamorph by creating constant intensity thresholds above which cells were counted as positively stained. Two independent observers tallied the number of neuronal profiles positively stained for both the specific and pan-sensory marker in each tissue section. The results from both observers were averaged and used to generate percentages of sensory neurons staining positively for individual sensory markers. For each marker, counts from at least five tissue sections per animal were averaged. 3 animals per genotype were analyzed and genotype averages were compared. For hindpaw staining, deeply anesthetized male mice were transcardially perfused with PBS for 4 minutes, plantar skin from the hindpaw was dissected out and post-fixed in 4% PFA for 18 hours before being cryoprotected in 30% sucrose. Tissue was embedded in OCT mounting media and stored at 280uC until sectioning. 20 mm sections were sectioned serially on a cryostat and stored at 220uC until needed. For TRPV1 hindpaw staining, mice were given a plantar injection of 1 mg of NGF (in 10 ml saline) 48 hours prior to transcardial perfusion. Further tissue Type III Nrg1, TRPV1 and Pain Sensation preparation was as described above, except for the use of 40 mm cryostat sections. For immunofluorescent staining, sections were rehydrated in PBS-T and blocked in 10% NDS in PBS-T for 1.5 hours at room temperature before primary antibodies were applied overnight at 4uC. Primary antibodies used to stain the hindpaw include those against CGRP (rabbit, 1:1000, T-4032 Bachem), TRPV1 (guinea pig, 1:1000, GP14100 Neuromics) and Type III Nrg1 (chicken, 1:2000). The following day, secondary antibodies conjugated to Alexa488 or Alexa546 (1:500, Molecular Probes) (diluted in 10% NDS in PBS-T) were applied and sections were coverslipped with Vectashield (containing DAPI) and sealed with nail polish. For immunofluorescent staining of primary DRG cultures, cells grown for 36–48 hours were rinsed twice in PBS, fixed in 4% PFA, permeabilized in PBS-T for 10 minutes and blocked in 10% NDS in PBS for 1 hour at room temperature before incubation with primary antibody. To assess activation of PtdIns3K along WT and Type III Nrg1+/2 axons, cultures were serum-starved for 4 hours, and then treated with either sErbB4-ECD (300 ng/ml) or NGF (100 ng/ml) for 15 minutes at 37uC before being stained with antibodies against pAKT (Ser473, rabbit, 1:200, 4060 Cell Signaling Technology), Type III Nrg1 (chicken, 1:2:000) and pan-axonal proteins (mouse, 1:500, SMI-312R Covance) over- night at 4uC followed by secondary detection with Alexa488-, Alexa594- or AMCA-conjugated secondary antibodies (diluted in 10% NDS in PBS). Fields of view containing axons selected based on their expression of Type III Nrg1 and pan-axonal protein were imaged using epifluorescence, and photographed with a constant exposure time using an Axio Imager microscope (Carl Zeiss, Inc.) equipped with a 636 oil Plan-Apochromat objective (1.4 NA), a charge-coupled device camera (Hamamatsu), and Metamorph software (MDS Analytical Technologies). At least 5 fields of view were acquired from each of the three animals per genotype. Briefly, axons were outlined in Metamorph using the pan-axonal marker as a guide, the outlined regions were transferred to the pAKT image, and the average fluorescence intensity (AFI) of the pAKT staining along these regions was recorded. Intensity of pAKT staining was averaged by animal within a given treatment and average pAKT intensity was compared by genotype and treatment. To quantify levels of Type III Nrg1 along P21 WT and Type III Nrg1+/2 axons, cultures were treated with antibodies that recognized either an extra-cellular epitope of Type III Nrg1 (chicken, 1:2000, 2 hours at 37uC) or the C-terminal intracellular domain (Nrg1-ICD, rabbit, 1:200, sc348 Santa Cruz, overnight at 4uC). Cultures were also stained for peripherin (rabbit or mouse, 1:1000), before secondary detection with Alexa488- or Alexa594- conjugated secondary antibodies (diluted in 10% NDS in PBS). Fields of view containing axons (identified based on expression of peripherin) were imaged using epifluorescence using a 636 oil Plan-Apochromat objective (1.4 NA) and photographed with a constant exposure time. At least 5 images were acquired from each of the three animals per genotype. Images were processed with MetaMorph software. Axons were outlined using peripherin staining as a guide, and the number of Type III Nrg1+ and Nrg1-ICD+ punctae along these peripherin+ axons with intensity greater than a defined threshold value were quantified and expressed as the number of punctae per 100 mm of axon length. These values were averaged within a given image and compared by genotype. Calcium Imaging At 3 days in vitro, primary DRG cultures were loaded with 5 mM Fluo-4 Ca2+ binding dye (Invitrogen) in 16 HEPES PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e25108