The choice of a non-viral neuro-tracer depends on several factors including its labeling efficiency and uptake mechanisms [12,13]. Several molecules such as carbo-cyanine dyes, beads, dextrans, lectins, trophins, amino acides, inorganic fluorescent molecules and bacterial toxins have been previously used as non-viral neuro-tracers. Among these several options, inorganic fluorescent molecules have key features of an ideal neuro-tracer, which include its selectivity, efficiency and availability for multi-color labeling [14]. Important factors to be considered when choosing a neuro-tracer are: a) duration of time to label the target after dye application; b) duration of fluorescent labeling after uptake (how long the fluorescent labeling lasts); and c) labeling efficiency of the candidate neuro-tracer which is determined by number of labeled cells and intensity of labeling. The application methods for neuro-tracers include there main methods of dye delivery: pressure injection, iontophoretic injection, and crystal powder application [13]. The current paper describes a detailed protocol for application of FB by pressure injection method using a 27 to 32 gauge metal needle inserted into a 25 μL Hamilton syringe or crystal powder application method.

The time required for the dye to label nerves and neurons innervating a certain tissue/organ varies significantly and depends on the type of the chosen neuro-tracer and structure of the targeted organ undergoing dye injections. In addition, a number of other factors should be considered. Animal species and a length of neuronal projection from the target organ (e.g., kidney) to neuro-tracer’s final destination (e.g., DRG neurons) are additional critical factors influencing the time of the labeling period. The time period sufficient for a neuro-tracer to travel along the nerves and provide consistent labeling outcomes has been previously investigated and discussed [15]. For example, FB was reported to produce some labeling as early as 2 days after injections with maximal labeling usually seen at 1–2 weeks. Table 1 summarizes available reports showing the choice of species and length of neuronal projection on the labeling periods of FB in each study [16–21]. Once the window of the labeling period is determined, the end point for tissue collection can be chosen based on the nature of the experiments planned to be done with the labeled target, as well as subsequent experimental interventions. For instance, Wang et al [8] studied functional interactions between P2Y receptors and transient receptor potential vanilloid type 1 (TRPV1) in kidney projecting sensory neurons. The group used a rat model, and applied the fluorescent tracer to the renal artery to label sensory neurons located within DRG neurons. Since they planned to proceed with immunohistochemial studies and electrophysiological recordings, 6 days of labeling period was sufficient to label sensory neurons in their study. A study by Malykhina et al [10] performed a dual-labeling procedure using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyaine perchlorate (DiI) injected in the colon and FB in the urinary bladder in adult rats 3 to 5 days in advance before inducing trinitrobenzene sulfonic acid (TNBS) colitis in their animal model. This approach allowed sufficient time for retrograde labeling to occur, and animal model to develop experimental colitis at the same time.

Materials and reagents required for each step are listed below in alphabetical order. Alternatives can be considered for each user’s convenience.

Items marked with asterisks are from World Precision Instruments, Sarasota, FL, USA.

Surgical procedure to inject a neuro-tracer should be performed in a sterile setting. The method and duration of anesthesia, as well as postoperative care should be discussed with veterinarian and approved by the Institutional Animal Care and Use Committee (IACUC) at an affiliated institution in the United States, or by other professional affiliates in the countries outside of the United States. During the surgical procedure, depth of anesthesia, animal homeostasis and recovery from anesthesia should be closely monitored. Postoperative care should include management of post-operative pain and all other potential post-surgical complications, administration of analgesics/antibiotics, and maintaining the records of post-operative care.

Here we outline the details of the surgical procedure to inject the retrograde neuro-tracer FB in the kidney. The mouse is anesthetized according to the approved animal protocol, and the depth of anesthesia should be confirmed by hind paw pinch to check plantar reflexes. Bu-prenorphine is administered by subcutaneous injection at a dose of 0.05–0.1 mg/kg prior to surgery for analgesia. The mouse is placed on its right lateral side, the fur is shaved, and the surgical area is prepped with providone-iodine. The kidney is exposed through a skin incision in the left flank. The neuro-tracer FB is applied by one of two methods. For the direct injection to the kidney, a total volume of up to 40 μL of 4% (weight/volume) of FB in 0.9% saline is divided into six to eight injections using a 27–32 gauge metal needle inserted into a 25 μL Hamilton syringe. For crystal powder application method, powdered FB crystals are placed on the surface of the kidney. To prevent neuro-tracer spillage, the application site is swabbed with dry cotton tip applicators after the procedure, followed by cleaning the application site and adjacent organs with PBS-soaked gauze squares. The abdominal wall, muscles and skin are readjusted and closed with 4–0 PDS suture and skin staples. Buprenorphine at a dosage of 0.05–0.1 mg/kg should be administered subcutaneously every 6 to 8 hours for 24 hours total to relieve post-operative pain.

When a neuro-tracer is injected at the application site, the leakage of neuro-tracers onto adjacent organs may occur resulting in undesired labeling [17]. Therefore, it is crucial to prevent cross-labeling of tissues during the application process, as well as to exclude any off target labeled tissues from further analyses. Precautions necessary to avoid neuro-tracer spillage have been described in the previous literature [7,10,22,23], and should be taken during the surgical procedure, tissue harvest and sectioning of the target tissues.

First, a minimal but sufficient amount of a neuro-tracer should be applied to the intended application site. It should be noted that a greater amount of the neuro-tracer may increase the chances of leakage and unwanted labeling of surrounding tissues. On the other side, a lesser amount of the neuro-tracer may result in deficient labeling of neuronal targets. Therefore, optimization of the tracer amount is essential for successful labeling of kidney innervation. In case of dual or multi-organ labeling, each neuro-tracer should be applied using a separate syringe (for the injection method) or forceps (for the crystal powder method) [22]. For the injection method done by using either a pressure-injection system or a manual plunger, the injection should be made as slowly as possible, and the needle should be left inserted for a minute in the tissue to prevent the leakage of the dye [10,24]. An absorbing material like gauze is recommended to be used to isolate the surgical site during dye injections to prevent the spillage [8,10]. After injection, the application site should be swabbed by dry cotton tip applicators to soak up residual neuro-tracers [11,17]. Then both the application site and adjacent organs are swabbed with PBS-soaked cotton tip applicators. Separate cotton tip applicators should be used for the application site and adjacent organs to prevent cross-labeling of the tissues. Given that neuro-tracer uptake by target organs can stain the site of neuro-tracer uptake [10], errant labeling in adjacent organs can be revealed by using a dissecting microscope during the process of tissue harvest. Lastly, errant labeling should be identified by examining sectioned slides of both target and adjacent organs under a fluorescent microscope. Tissue slides with errant labeling should be excluded from further analysis [7,17] (Fig. 1).

On the day when the labeled ganglia/neurons are collected, it is strongly recommended that the target organ injected with dyes and adjacent organs are collected for potential spillage of the dyes in vivo after the surgery. For example, the kidney, spleen, pancreas and stomach can be considered for collection. If there is any other tissue that may have been affected by neuro-tracer spillage, that tissue should be collected as well for further analysis. Please note that by keeping the orientation of the organs for dissection as close to their original way as possible, the location of neuro-tracer spillage may be more easily identified. After the dissection, all tissues are washed with PBS and fixed in 4% PFA in phosphate buffer (0.1 M, pH 7.4) overnight at 4°C. They are then transferred to 20% sucrose in phosphate buffer for at least 48 hours at 4°C for cryoprotection. For cryosectioning at −20°C, tissues are immersed in Tissue-Teck O.C.T Compound and frozen at −20°C. The organ blocks are cut into 10 μm serial sections using a cryostat. At least 5 sections per each tissue should be collected for analysis. The slides could be imaged under the fluorescent microscope to select the tissues with and without labeling.

DRG neurons from an adult mouse can be dissected either by spinal column isolation [20] or open access from the dorsal side of the spine. The former procedure is described in depth by Sleigh et al [25]. Briefly, the pelt is pulled up and fully removed to expose the musculature followed by spinal column isolation. Each spinal segment can be identified by using two methods. First, as Sleigh et al [25] suggested, the most caudal ribs can be labeled with a fine marker pen; thoracic segment (T) 13 is found just caudal to the ribs. Second, the spinal column can be harvested with ribs intact, which allows the spinal segments to be counted. Extraneous muscle, fat, nerves and other soft tissues are removed. Then DRG neurons can be harvested with or without transverse excision of the spinal column by pulling out dorsal root nerves with caution.

The other way to dissect DRG neurons is to gain open access to the spinal cord from the dorsal side of the spine. The euthanized mouse is usually decapitated to drain the blood. The dorsal pelt is removed to expose the musculature, and the access to the spine is gained via the cervical segments followed by the opening of the dorsal spine all way down to the lumbar segments. Spinal nerves along with the spinal column are pulled aside making DRG neurons visible for isolation. DRG neurons are processed the same way as the other tissues for subsequent analyses, as described above. However, the blocks of DRG neurons should be cut into 10 μm serial sections with all sections or every other section to be collected (preferable) due to small diameter of the ganglia.

Unless harvested tissues are designated for cell culture and expression profile assay, cardiac perfusion with fixatives is highly recommended prior to the tissue harvest. Rapid preservation of the tissue using fixative provides rigidity for many tissues including the kidneys. Please note that cervical dislocation should not be considered as an option for euthanasia to isolate the neural tissues because the procedure may damage DRG neurons and other neural structures. A detailed protocol and video clip for intracardial perfusion are available at http://www.jove.com/video/3564/whole-animal-perfusion-fixation-for-rodents [27].

Briefly, the mouse should be fully anesthetized, and the anesthetic effect should be confirmed by hind paw pinch to check plantar reflexes. The chest is then opened via a 5–6 cm lateral incision just beneath the rib cage. The diaphragm is carefully opened via a small incision using the curved, blunt scissors until the pleural cavity is exposed. The lung is displaced and the rib cage is incised up to the collarbone on both the ipsilateral and contralateral sides. The sternum is lifted, and any connective tissue surrounding the heart is removed. The posterior end of the left ventricle is located and pierced using iris scissors. A 15-gauge blunt or olive-tipped perfusion needle is inserted in the right atrium to allow blood exit the vasculature. After the blood draining, the catheter is perfused with PBS first followed by slow perfusion with 4% PFA for in vivo fixation of the tissues.

In general, FB provides reproducible and robust labeling outcomes [12,16]. The best efficiency of FB uptake is achieved by following the manufacturer’s direction for storage conditions and dissolving method. Even if the uptake of neuro-tracers occurs properly, labeled cells can fade out after the sections are placed on slides. This can be prevented by using anti-fade mounting media fluorogel. In addition, minimal duration of light exposure during imaging may help with the loss of fluorescent signal. It was previously discussed that combination or sequential application of different neuro-tracers may result in deficient labeling [12]. However, both FB and DiI are known to provide a long-lasting efficient labeling, and are considered to be the efficient dyes (Fig. 4).