Given sensitivity and species detection issues inherent in our available NET-directed antibodies, we sought to generate a monoclonal antibody against the mouse NET (mNET) suitable for rodent-targeted Western blots, immunoprecipitation studies, and immunocytochemistry. We selected a sequence in mNET (amino acids 5–17) based on predicted antigenicity, and lack of conservation with other monoamine transporters such as the serotonin transporter (SERT) and dopamine transporter (DAT). This peptide is found in the N-terminal intracellular domain. Among NET paralogs, however, the mNET sequence chosen is highly conserved, differing from the rat NET (rNET) at only one amino acid (Figure 1A). Figure 1A provides an alignment of the NET amino termini from several species as well as the orthologs DAT and SERT from mouse, rat and human [57,58]. BLAST searches of mammalian protein databases yielded no significant matches to the epitope except those found in NET paralogs (data not shown). Following immunization with the coupled peptide (see Methods), we developed multiple hybridoma cell lines that produced antibodies yielding positive signals in peptide-based ELISA assays (data not shown). From these data, we selected a hybridoma (NET-05) for further characterization and use it throughout the experiments presented in this report.

Immunoblots using cell lysates from COS cells expressing rNET demonstrate that NET-05 recognizes rNET despite the one amino acid change relative to the mouse NET peptide (Figure 1B). However, NET-05 fails to detect human NET (hNET), which differs from mNET at two consecutive amino acids (Figure 1B). To verify whether NET-05 recognizes mNET in native tissue, we prepared post-nuclear fractions (S1) from wild type mouse cortex and from mice with mNET genetically deleted (NET KO). [28,59]. Our blots identify a ~70 kDa band present in wildtype but not NET KO samples (Figure 1C). These results demonstrate that NET-05 recognizes rodent NETs via Western blots of extracts from both heterologous expression systems and native tissues.

The distribution of noradrenergic neurons and processes in rodent brain is well-known [4] and the distribution of NET has been previously described in these pathways using polyclonal antisera [49,53,60]. To verify the utility of NET-05 for immunocytochemistry, we assessed the staining pattern obtained using NET-05, marking catecholamine neurons in parallel using anti-tyrosine hydroxylase (TH). As shown in a low magnification view (10×) of the locus ceruleus (LC), NET-05 immunoreactivity colocalizes extensively with staining for TH (Figure 2A–C) and extends into dendrites (arrow). Higher magnification (63×) views are shown in Figure 2D–F. NET-05 and TH staining similarly colocalize in the bed nucleus of the stria terminalis (BNST), an area rich in NE axons and varicosities (Figure 2G–L) [61-64]. NE fibers are more dense than in the ventral BNST (vBNST) as compared to the dorsal division (dBNST) [61-64]. Confirming this pattern, we observed a higher density of NET fiber labeling in the vBNST (Figure 2J–L) as compared to the dBNST (Figure 2G–I). Inset panels of Figure 2G–L using a stack from multiple confocal sections (n = 6) demonstrate NET labeling along individual fibers. Both TH and NET antigens appear enriched in punctate domains along individual fibers, presumably the well-known beaded varicosities that comprise NE release sites [4]. In these fields, some fibers are TH positive but show no NET immunoreactivity (see inset Figure 2I). These fibers are most likely dopaminergic fibers that express TH but lack NET and which are known to innervate this region [65]. To demonstrate specificity of the NET-05 reagent, we stained vBNST sections from wildtype and NET KO mice (Figure 2J–O). Although the pattern of TH staining in sections from NET KO (Figure 2M) appeared similar to that from wild type mice (Figure 2J), no NET staining was evident in the NET KO (Figure 2N). Similar negative staining results were obtained with sections incubated without primary antibody or pre-absorbed with peptide (data not shown).

Having validated the utility of NET-05 antibody for detection of mouse brain NET in multiple assays, we examined the hypothesis that NET is sorted to dense core vesicles that harbor VMAT2. Figure 3 illustrates the distribution of NET (Figure 3A) and VMAT2 (Figure 3B) immunoreactivity in the mouse hippocampus, focusing on the NE rich hilus of the dentate gyrus. Labeling of NET in these fields is similar to that achieved with polyclonal NET antisera [49,53]. Although colabeling was evident in most neuronal varicosities, colocalization was not uniform as some NET-positive elements displayed low levels of VMAT2 immunoreactivity and some varicosities appeared to have high levels of VMAT2 relative to NET. Regardless, staining in the hippocampus, as in the LC and BNST, is absent in the NET KO (data not shown), confirming NET-05 specificity. These light-level immunocytochemical data confirm expression of NET at discontinuous axonal in the CNS and indicates that the relative abundance of NET and VMAT2 differs across individual fibers.

Although we achieved evidence of specific NET labeling within or on neuronal membranes, the small size of CNS NE varicosities, unfortunately, precluded quantitative immunocytochemical analysis of NET distribution. Previously, we observed that NET is enriched in sympathetic varicosities in situ [43,53]. Therefore, we continued our studies using SCG neurons that elaborate profuse noradrenergic fibers in culture [49,53]. Furthermore, SCG preparations present large varicosities extending laterally from axonal membranes (for example see 8A). As with the CNS preparations described above, we verified the specificity of NET-05 in the SCG cultures and then examined NET subcellular distribution in relation to multiple markers of vesicles and membrane subdomains. Figure 4 shows single confocal sections and corresponding DIC images of cultured SCGs focused on either the cell bodies (Figure 4A–C) or the processes (Figure 4D–F). In the cell bodies, NET labeling surrounds the nucleus in a pattern consistent with Golgi compartments. A low level of immunoreactivity was associated with the plasma membrane, most evident at sites where cell bodies contact one another (Figure 4B). Importantly, NET-05 labeling exhibited a punctate pattern throughout SCG processes (Figure 4E). To demonstrate specificity of NET-05 labeling in this preparation, we stained SCG cultures prepared from NET KO mice (Figure 4G–L). As shown in Figure 4H and 4K, the KO neurons lack NET staining.

Next, we compared the distribution of NET as detected with NET-05, which requires permeabilization, to that of NET 43408, a polyclonal NET antibody that targets the NET 2^nd extracellular loop and which therefore reports NET expression at the cell surface when used in the absence of detergent [53]. Whereas NET-05 detects transporter throughout neuronal processes (Figure 4E), NET 43408 reveals NET surface expression particularly evident at boutons (Figure 4N, arrows, for higher magnification, see Figure 8a–d). These boutons store NPY (see below) and thus may represent one class of NE neurons that innervate smooth muscle [66,67].

The studies above indicate that the NET-05 labeling associated with SCG neurites, in the presence of detergent, represents largely intracellular pools of transporter-containing membranes, whereas boutons are the primary sites of surface NET expression. To test if intracellular NET resides on large dense core vesicles, we double stained SCGs for NET and NPY (a neuropeptide sorted to large dense core vesicles in SCGs [66,68]. As indicated above, NET-05 labeled the surface and intracellular membranes of single SCG boutons (Figure 5A, B). NPY labeling was enriched in the cytoplasm of boutons and relatively low in abundance within processes, relative to that observed for NET (Figure 5C). Importantly, we observed no colocalization with NET in boutons (Figure 5A–D see arrows and Figure 5D inset) suggesting NET is not sorted to large dense core vesicles (LDCVs). We considered the possibility that bouton resident NET vesicles might represent small dense core vesicles (SDCVs) which originate at presynaptic sites. Upon fusion with the plasma membrane, LDCVs release NPY and LDCV membrane proteins, including VMAT2, are endocytosed to form SDCVs [52,55,56,69]. Just as we observed for NPY, VMAT2 labeling was also enriched in the cytoplasm of SCG boutons and in relatively low abundance in processes as compared to NET (Figure 5G). Importantly, within the bouton of double labeled neurons, VMAT2 displayed little apparent colocalization with NET (see arrows and Figure 5H inset). Additionally, we found little if any colocalization of synaptophysin, which labels an endocytic compartment through which SDCVs [52,56,70] (Figure 5I–L, 5L inset). Together these findings support the contention that in SCG boutons, NET is not sorted to any class of dense core vesicles.

The evaluations presented above of NET colocalization with VMAT2 and other proteins are qualitative, based on visual inspection of chromatic changes in labeling when using multiple fluorophore-labeled secondary antibodies. To investigate colocalization quantitatively, we plotted the normalized intensities for NET and VMAT2 (Figure 5M upper left plot) or NET and synaptophysin (Figure 5N upper left plot) in both axonal regions lacking varicosities as well as in boutons, and found no evidence of correlation. To achieve a quantitative evaluation of colocalization, we implemented an unbiased analytical method that has been validated for determination of protein colocalization in intact cells [71]. Stanley's group reasoned that if two proteins are on the same organelle or interacted, then the paired pixel intensities should vary in synchrony over space [71]. This method, termed Intensity Correlation Analysis (ICA) plots the normalized pixel intensity for a particular protein (e.g. NET) in relation to the normalized intensity of the other protein (e.g. VMAT2). The quantitative index produced in the ICA analysis is termed the Product of the Difference from the Mean, PDM [71]. In graphical form, the normalized paired pixel intensity of each protein (e.g. NET) is plotted against the PDM of the two proteins of interest, producing a distribution of points between the values of 0 and 1 on the y axis and between -0.5 to +0.5 on the x axis (e.g. Figure 6M and 6N bottom panels). Pixel intensities that indicate colocalization are associated with positive values of the x axis. For statistical analysis, the number of pixels with a positive × value is divided by the total number pixels. This procedure yields the intensity correlation quotient (ICQ) which can be analyzed by a nonparametric sign test [71]. Analysis of NET and VMAT2 yielded a nonsignificant ICQ value (P > .05) consistent with NET and VMAT2 as localized to different membrane compartments. The ICQ value for NET and synaptophysin in axons is low, though a significant association was identified (P < 0.001) (see arrow in Figure 5L).

In Figure 5M and 5N, we present the spatial distribution of PDM values (upper right panels) using a pseudocolored scale. Pixels with positive PDM values are in orange (high colocalization) and negative PDM values in purple (segregation). Below this display, we present the standard two-color merge of the same region of the axon. Whereas NET and VMAT2 show no extensive regions of colocalization, for NET and synaptophysin, limited regions within the axon appear to demonstrate positive colocalization (Figure 5, thick arrows). However, when we performed these analyses in boutons, we detect no evidence of NET colocalization with VMAT2, NPY, or synaptophysin (data not shown), further confirming segregation of NET from LDCVs in axons as well as SDCVs in boutons.

To evaluate the distribution of NET and VMAT2 via an alternative approach, we performed immunoisolation experiments of SCG membranes. To establish the utility of NET-05 for immunoprecipitations, we incubated antibody with detergent extracts of cortical synaptosomes and probed NET with the same antibody (Figure 6A). Immunopreciptated NET migrates at ~70 kDa in total extracts (Figure 1), migrating on SDS-PAGE just above the heavy chain of NET-05 IgG (52 kDA) (see Figure 6A; beads alone). Additionally, we also immunoprecipitated intact organelles from hypotonic lysates of mouse cortical synaptosomes (prepared in the absence of detergent). The 70 kDa NET band observed in wildtype (WT) but not NET KO lysates demonstrates specific NET recovery (Figure 6B). In Figure 6C, we present results of organelle isolation from cultured SCGs using NET-05, VMAT2 or Na/K ATPase antibodies for membrane isolation, followed by blots of NET and VMAT2. NET-05 isolated membranes contained only background levels of VMAT2 (comparable to that obtained with Na/K ATPase coated beads). Conversely, VMAT2-coated beads recovered negligible, background levels of NET proteins. Antibody-free beads yielded negligible background NET isolation. These findings support the contention derived from staining experiments in SCG cultures that NET and VMAT2 are segregated to distinct membrane compartments.

Recently, SCAMP2 has been identified as an N-terminal SERT and DAT interacting protein, with suggestions of a role in transporter sorting or trafficking [72]. It seemed likely that SCAMP2 might also associate with NET as well, and thus we explored NET and SCAMP2 colocalization in SCG cultures (Figure 7A–D). SCAMP2 immunoreactivity was readily detected in both axons and boutons. In boutons, we observed no statistically significant colocalization with NET (Figure 7A–D). Likewise, neither the PDM image of a representative axon nor the two color merge of the same segment (Figure 7I, top right) gives evidence of strong colocalization between NET and SCAMP2. The ICA plot (Figure 7I bottom) appears symmetric with a "cloud" of points in the lower quadrant. Nonetheless, the ICQ value (+0.027) reflects a significant association (P = 0.01) that supports the possibility that a small fraction of NET resides on SCAMP2-positive organelles or that a minor fraction of SCAMP2 resides on NET-transport vesicles.

We have previously demonstrated that NET colocalizes with syntaxin 1A in sympathetic varicosities in situ [43,49,53]. Indeed, we gained clear evidence in the SCG cultures for extensive colocalization of syntaxin 1A and NET (Figure 7E–H) at the surface of boutons. Since it is unknown whether NET and syntaxin 1A are sorted to distinct axonal transport vesicles, we examined the correlation of these two proteins in axons. Visually, we observed apparent colocalization of NET and syntaxin 1A. Indeed, the distribution of pixel intensities for axonal NET and syntaxin 1A (Figure 7J top) demonstrates a positive spatial correlation for this labeling. ICA/ICQ analysis confirms a significant association of axonal NET and syntaxin 1A (P ≤ 0.001). When a similar analysis was performed in boutons, again the two proteins exhibited a highly statistically significant colocalization (data not shown), consistent with previous biochemical studies [43]. These findings indicate that NET is sorted to syntaxin 1A containing vesicles such that both SNARE protein and transporter can be deposited at the plasma membrane upon membrane fusion.

Plasma membranes are not homogenous lipid bilayers but rather contain discrete subdomains rich in cholesterol and sphingolipids termed lipid rafts [73,74]. Interestingly, biochemical fractionation and pharmacological studies of placental trophoblasts demonstrate that a portion of NETs exists in lipid rafts [46]. Whether neuronal NET localizes to these domains has not been established. Cholera toxin-B (CTx-B) targets cell-surface localized GM1 gangliosides that are enriched in lipid-rafts [75]. We used fluorescent CTx-B to label either nonpermeabilized or detergent-permeabilized SCGs to determine if NET localizes to lipid raft compartments. As shown in Figure 8A–D, fluorescent CTx-B labeling of SCG processes and boutons under nonpermeabilized conditions was nonuniform as expected of lipid raft membrane subdomains. Remarkably, all sites labeled with CTx-B under nonpermeabilized conditions displayed NET05 immunoreactivity (Figure 8A–D). However, the converse was not true, demonstrating that NET in the plasma membrane of boutons localizes to both raft and non-raft membranes. In permeabilized conditions, CTx-B labeling of processes revealed little colocalization with NET (Figure 8E–L, see thicker arrows). Consistent with these observations, the spatial distribution of pixel intensities for NET and CTx-B (Figure 8M top) demonstrated a positive correlation for nonpermeabilized boutons whereas axonal labeling under permeabilized conditions exhibited a random pattern is observed (Figure 8N top). ICA/ICQ analysis yielded a significant association of NET and CTx-B in nonpermeabilized (ICQ = 0.315; P < 0.001) as compared to permeabilized axonal preparations (ICQ = -0.032; P > 0.05) (Figure 8M and 8N). These findings support the targeting of NET proteins to lipid rafts once transporters have been inserted into the presynaptic membrane.

We and others have developed polyclonal antisera capable of detecting NET protein by immunocytochemical techniques in situ [49,53,60,84,85]. Prior to this study, we had also developed a high-affinity, mouse monoclonal antibody that targets human NET (hNET) [86]. A monoclonal antibody suitable for colocalization studies with other polyclonal reagents in rodent preparations and sensitive enough for detection of native protein by immunoblots was not available, prompting our current efforts. We thus generated the mouse NET amino terminal peptide RMNPQVQPELGGA (amino acids 5–17) via solid-phase techniques (Tufts University Core Facility), adding a C-terminal cysteine to facilitate coupling to keyhole limpet hemocyanin (KLH). Subsequent efforts using animals for antibody development and characterization were performed with attention to the NIH Guide for the Care and Use of Laboratory Animals under protocols approved by Institutional Animal Care and Use Committees of the Vanderbilt University School of Medicine. Mouse monoclonal antibodies to the NET peptide conjugate were prepared following standard hybridoma techniques [87] in collaboration with MAb Technologies (Mabtechnologies.com). Briefly, 3 BALB/c mice were given subcutaneous and intraperitoneal primary injections with the KLH conjugate emulsified in Freund's complete adjuvant. Subsequent booster shots were given subcutaneously at 3–4 week intervals until a strong immune response to the antigen was produced, as assessed via ELISA assays on tail vein blood. The best responder was sacrificed, the spleen was isolated and spleen cells were extracted and fused to a mouse myeloma cell line (P3X63.Ag8.653) using polyethylene glycol (PEG). Cells were plated in 8 × 96 well plates under hypoxanthine-aminopterin-thymidine (HAT) selection, supplementing with peritoneal macrophage feeder cells. We screened the culture medium from individual wells for the presence of antigen secreting cells, as assessed by ELISA assays as well as Western blots of mouse and rat NET transfected cells. Cells from wells that tested positive were cloned by limiting dilution in 96 well plates. Hybridomas in positive wells from the cloning plates were again re-cloned by limiting dilution to establish a final sub-cloned antibody producing hybridoma cell line (NET-05). Isotype analysis revealed the immunoglobulin secreted to be of the IgG_2b subtype.

To evaluate of the sensitivity and specificity of NET-05, we compared results to our hNET-specific monoclonal antibody (NET17-1; MAb Technologies, 1:1000) [86,88] as well as a rabbit anti-NET polyclonal antiserum, designated 43408 [53]. The antigen for NET 43408 is a KLH conjugated peptide derived from the transporter's 2^nd extracellular loop. This anti-NET polyclonal antiserum (43408) labels mouse noradrenergic neurons in the CNS and periphery that is lost with preparations from NET KO mice [53]. Additional commercially available reagents used included anti-tyrosine hydroxylase (TH, Chemicon, 1:3000); anti-NPY (Santa Cruz 1:200), anti-vesicular monoamine transporter 2 (VMAT2 Chemicon and Santa Cruz 1:1000), anti-synaptophysin (synapto, Boehringer Mannheim, 1:300) and anti-dopamine beta hydoxylase (DBH, Chemicon 1:300). Anti-SCAMP2 was gift of Dr David Castle (University of Virginia). Secondary antibody reagents included donkey anti-rabbit IgG conjugated with Cy3 or Cy2 (Jackson Labs, 1:1000), donkey anti-mouse IgG conjugated with Cy2 (Jackson Labs, 1:500), goat anti-rabbit conjugated with Alexa 568 (Molecular Probes, 1:1000), goat anti-mouse conjugated with Alexa 488 or Alexa 568 (Molecular Probes, 1:1000), goat anti-rmouse IgG2_b conjugated with Alexa 488 (Molecular Probes, 1:1000) and goat anti-rmouse IgG1 conjugated with Alexa 568 (Molecular Probes, 1:1000). Alexa Flour 488 conjugated cholera toxin subunit B was from Molecular Probes.

Western analysis and immunopreciptations were performed as previously described [35,43,54]. Briefly, cortical synaptosomes were prepared [89,90] and either lysed in 1% Triton X-100 for one hr or lysed in hypotonic buffer [89,91]. Membranes from cultured superior cervical ganglion neurons (SCGs) were also prepared by hypotonic lysis to release synaptic vesicles and other cytoplasmic organelles as in Nagy et al and modified by Huttner and coworkers [89,91]. This method involves a step in which membranes are incubated with buffered water allowing release of intact synaptic vesicles [89,91]; however, larger organelles may be lysed. Detergent extracted membranes (1 mL) were cleared of non-solubilized membranes by a 30 minute centrifugation at 100,000 × g and 10 uL NET-05 was added. After a 2 hr incubation, extracts were incubated with Protein A beads (Sigma) for 1 hr. Beads were washed and eluted with Laemmli buffer [92]. Synaptosomes (1 mL) or SCGs lysed with hypotonic media were used after the addition of 150 mM NaCl and centrifugation at 1000 × g for 20 minutes [89]. Membranes were incubated for 4 hrs with 10 uL NET-05 and then incubated for 2 hr with anti-mouse paramagnetic beads (Dynal; Invitrogen). Beads were washed in buffer 5× and then eluted with Laemmli buffer prior to separation of proteins by SDS-PAGE and transfer to Polyvinylidene Difluoride (Biorad, PVDF 0.2 micron) for immunoblotting. NET was detected using NET-05 and immunoreactivity was observed using chemiluminescence (Western Lightning Plus-ECL, PerkinElmer) insuring exposures were conducted in the linear range of exposed X-ray film.

Adult (8–12 weeks) wild type male mice (C57Bl/6J) and age-matched NET KO mice [28,59] were used to examine CNS NET distribution whereas P0–P5 pups where used for the culturing of neurons from the superior cervical ganglion as outlined below. NET KO mice were generously provided by Dr. Marc Caron (HHMI, Duke University School of Medicine). Animals were housed in 14-h/10-h light/dark cycles with food and water ad libitum. Mice were deeply anesthetized with Nembutal (80 mg/kg), and transcardially perfused with saline containing 4% paraformaldehyde. Brains were removed and immersed in fixative for two to forty eight hours at 4°C and then cryoprotected in 30% sucrose overnight at 4°C. Thirty or forty-micrometer-thick floating frozen sections were cut into PBS using a sledge microtome (Leica) and then incubated sequentially with primary antibodies (room temperature, 4 hr or forty eight hours at 4°C) and secondary antibodies (room temperature, 2 hr or twenty four hours at 4°C). Sections were permeabilized with phosphate-buffered saline (PBS) containing 0.15% or 0.2% TX-100. Nonspecific labeling was blocked by incubation in PBS containing 4% bovine serum albumin and 0.15% NP-40 or PBS containing 4% donkey serum for 1 hr prior to incubations with antibody solutions containing 4% bovine serum albumin and 0.05% NP-40 or 0.1% TX-100 and 2% donkey serum. Controls included incubations in the absence of primary and secondary antibody reagents and as well as the use of sections prepared from the brains of NET KO mice. Immunofluorescence was visualized using a Zeiss LSM 510 Meta confocal imaging system equipped with internal He/Ne and external Ar/Kr lasers (VUMC Cell Imaging Core Resource, Sam Wells Director, supported by NIH Grants CA68485 and DK20593) with output at 488 (Cy2, Alexa 488), 568 (Cy3, Alexa 568) and 633 (Cy5, Alexa 633) nm. Z-series were collected by optical sectioning at intervals ranging from 0.25 to 1 μm depending on the magnification used, followed in some cases by 3D reconstruction. Image processing and montage assembly were performed using either Zeiss or Metamorph software and Adobe Photoshop. Designation of anatomical structures for dissection and immunostaining followed regional and nuclear designations of [93].

SCGs were cultured as previously described [43,53]. Briefly, ganglia from 0–5 day old wild type pups (C57Bl/6J) and NET KO pups were dissected and incubated for 30 min at 37°C in collagenase (3 mg/ml; Sigma) and trypsin (0.5 mg/mL/Gibco) followed by termination of digestion using 10% fetal bovine serum (FBS, Atlanta Biologicals) in UltraCulture medium (BioWhittaker). To purify SCGs cells away from fibroblasts, cells were incubated in UltraCulture medium supplemented with 3% FBS, nerve growth factor (NGF, 20 ng/mL; Harlan) and 2 mM L-glutamine for 2 h at 37°C, SCGs were removed, centrifuged for 5 min, resuspended in supplemented UltraCulture medium and then transferred onto poly-D-lysine and collagen-coated coverslips. After 24 h of incubation at 37°C, cultures were treated with 1 μM 5-fluoro-5-deoxyuridine (FdU; Sigma) and grown for 14–28 days in supplemented Ultraculture medium prior to examination.

Colocalization of NET with plasma membrane and vesicular proteins was achieved on multiple, randomly selected fields of each preparation as previously described [49,53]. Fields from 10–30 per sample were used for each antibody. For axonal distribution analysis, regions of SCG processes were chosen that did not contain varicosities. Intensity values were measured using NIH Image and normalized to the peak intensity value. Paired pixel intensities were plotted as a scatter plot (Prism; GraphPad). The data were used to create the intensity correlation analysis plots and quantified by the intensity correlation quotient [71]. The ICA/ICQ method is based on covariance of signal intensities from two fluorophores in space and is evaluated via the nonparametric sign-test analysis [71]. The normal approximation of the sign test was used to test if these values were significantly different from 0 [71]. Alternatively, data were analyzed by one-sample t-test comparing means versus a value of 0. Multiplying the difference of staining intensity (A) at a particular pixel from the mean intensity (a) for that fluorophore across the analyzed space by the corresponding quantity for the other fluorophore (B-b) at the same pixel yields the Product of the Differences from the Mean [PDM = (A-a)(B-B)] for that pixel. Positive PDM values arise when the staining intensity of both are above the mean or are both present at high values below the mean. ICQ values were calculated first by determining the ratio between the number of positive PDM values and the total number of pixel values. From this ratio, 0.5 is subtracted to yield ICQ values distributed between -0.5 and +0.5 where random colocalization gives an ICQ of ~0, segregated or asynchronous colocalization gives 0 > ICQ ~-0.5, and dependent or synchronous colocalization yields 0 < ICQ ≤ + 0.5 [71]. PDM images were created using a plug-in for ImageJ found at .