The long propriospinal neurons (LPSNs) and short propriospinal neurons (SPSNs) were labeled with the retrograde tracer Fluoro-Ruby (MW, 10,000; 10% Fluoro-Ruby in PBS; Invitrogen) 2 weeks before the animals were perfused. Tracer injection procedures were modified slightly from a previous study34 (link). Briefly, the animals were anesthetized and placed in a stereotaxic frame. To label the LPSNs and SPSNs, 0.6 µL/site or 0.4 µL/site Fluoro-Ruby was bilaterally infused into the spinal cords at vertebral level T12 or C7 (lateral, ±0.8 mm, depth of 0.9 mm), respectively.
Fluoro ruby
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluoro-Ruby is a fluorescent dye used in various biological applications. It emits a red-orange fluorescence when excited by light at specific wavelengths. The dye can be used to label and track cellular components or specific molecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
Fluoromount g, by Southern Biotech (1 mentions)
Spongostan, by Johnson & Johnson (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Gelfoam, by Pfizer (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Tetramethylrhodamine dextran, by Thermo Fisher Scientific (1 mentions)
Vt1200, by Leica (1 mentions)
Cm1950, by Leica (1 mentions)
Fluorogold, by Thermo Fisher Scientific (1 mentions)
Oregon green bapta 1 am, by Thermo Fisher Scientific (1 mentions)
Mni caged glutamate, by Bio-Techne (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Cholera toxin b subunit, by Merck Group (1 mentions)
Lucifer yellow dextran, by Thermo Fisher Scientific (1 mentions)
22 protocols using fluoro ruby
1
Retrograde Labeling of Propriospinal Neurons
2
Anterograde and Retrograde Tracing of Brainstem Nuclei
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The anterograde tracers used were a 15% solution of biotinylated dextran amine (BDA, Invitrogen) or a 15% solution of Fluoro-Ruby (FR, Invitrogen) in 0.01M phosphate buffered saline (PBS). A 2% solution of Fluorogold (FG, Fluorochrome, Denver, CO, USA) in saline was employed as a retrograde tracer. BDA was pressure injected through a glass pipette cemented onto the tip of a Hamilton syringe (150 nl per injection site, 2–3 injections per region) whereas FG or FR were injected iontophoretically by passing a current of 2.5–5.0 μA for FG and 11 μA for FR for 10–20 min using a 7 s duty cycle. The most caudal injections into the brainstem succeeded in labeling Sp5ic, but not Sp5c, which was inaccessible because it is located under thick neck musculature. All brainstem tracer deposits consisted of injections made at 2–3 depths separated by ~200 μm to ensure filling of the complete nucleus.
3
Multi-Label Fluorescent Tissue Imaging
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FG (Fluorochrome, Denver, CO) was used for widefield, 2PEM, and FLIM. A concentration of 2% FG (w/v in distilled water, pH 6.5) was used to minimize neurotoxicity, and employed for animal experiments and characterization of dye properties in acqueous solution. Fluoro-Ruby (10% w/v in distilled water, Invitrogen, Carlsbad, CA) was used for facial subnuclei labeling to demonstrate the utility of multi-labeling techniques incorporating FG. Tissue sections were mounted in Fluoromount-G (SouthernBiotech, Birmingham, AL). EasyIndex (Life Canvas Technologies, Cambridge, MA) was used for refractive index matching.
4
Retrograde Labeling of Regenerated Motoneurons
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The medial gastrocnemius motoneuron pool was pre-labeled with 2% fluorescent tracer
Fast Blue one week before sciatic nerve injury and repair as described previously21 (link). In order to identify the number of spinal motoneurons which had regenerated
axons through the nerve graft and fibrin conduits in the same animals the sciatic nerve
was identified and transected 10 mm from the distal graft end at 11 weeks after
transplantation. A small cube of Spongostan (Ethicon, Johnson & Johnson Medical,
Sollentuna, Sweden) drenched in 10% aqueous solution of fluorescent tracer Fluoro-Ruby
(Invitrogen, Fisher Scientific) was applied to the proximal stump of the transected
nerve and isolated from the surrounding tissue by a well from mixture of silicone grease
and Vaseline to prevent leakage. Two hours later the sponge was removed, the nerve was
rinsed in normal saline and the wound closed in layers. The animals were left to survive
for one more week to enable labeling of the neurons before the termination of the
experiment.
Fast Blue one week before sciatic nerve injury and repair as described previously21 (link). In order to identify the number of spinal motoneurons which had regenerated
axons through the nerve graft and fibrin conduits in the same animals the sciatic nerve
was identified and transected 10 mm from the distal graft end at 11 weeks after
transplantation. A small cube of Spongostan (Ethicon, Johnson & Johnson Medical,
Sollentuna, Sweden) drenched in 10% aqueous solution of fluorescent tracer Fluoro-Ruby
(Invitrogen, Fisher Scientific) was applied to the proximal stump of the transected
nerve and isolated from the surrounding tissue by a well from mixture of silicone grease
and Vaseline to prevent leakage. Two hours later the sponge was removed, the nerve was
rinsed in normal saline and the wound closed in layers. The animals were left to survive
for one more week to enable labeling of the neurons before the termination of the
experiment.
5
Tracing Nerve Pathways with Fluorescent Dyes
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Two months after the cross-link operation, tracers were injected directly into the distal parts of the nerve sheaths (n = 2). A bolus of 1 μL of 10% fluoro-ruby (Invitrogen, USA; Cat D1817) diluted with distilled water was injected into the facial nerve, and the same quantity of fluoro-emerald (Invitrogen, USA; Cat D1820) was injected into the hypoglossal nerve. Five days thereafter, the animals were killed by perfusion with 10% formalin in 0.1 M phosphate buffer (PB) at room temperature, and the brains removed. The brains were immersed overnight in 0.1 M PB at 4 °C ontaining 20% sucrose for cryoprotection, and then sliced into 40-mm-thick sections on a freezing microtome. The sections were mounted on gelatin-coated slides and cover slipped.
6
Retrograde Labeling of Motor Neurons
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In separate experiments, mice were anesthetized with 3% isoflurane/oxygen and the nerve branches to medial and lateral heads of the left gastrocnemius were exposed and transected. On the contralateral side the sciatic nerve was transected at the level of the mid-thigh. Motor neurons were retrograde labeled by placing sterile Gelfoam saturated with 10% FluoroRuby (Invitrogen) over the respective nerve stumps, suturing the Gelfoam in place and closing the incision sites for recovery. Postoperative analgesia was achieved by treating mice with Carprofen (5 mg/kg) daily for 3 d. Following schedule 1 cull, the lumbar regions of spinal cords were obtained by extrusion with 5 ml of PBS 7 d after the onset of labeling. Spinal cords were fixed overnight in 10% formalin at 4°C, rinsed in PBS and processed for optical clearing according to previously published work (Ertürk et al., 2012 (link); Žygelytė et al., 2016 (link)). Labeled motor neurons were imaged using an Olympus FV1000 confocal microscope with the z-step size set at 4.5 μm. Z-stacks were visualized using ImageJ and the total number of labeled motor neurons were quantified with the use of the cell counter plug-in function while scrolling through z-stacks.
7
In Vivo 2-Photon Guided Cell Labeling
8
Retrograde Tracing of Facial Nerves
9
Mapping Somatosensory Cortex in Rodents
10
Retrograde Labeling of Spinal Motor Neurons
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Four retrograde tracers were used for labeling the motor neurons in the spinal cord as follows: FG (Fluorochrome, Denver, CO, United States) was dissolved at 5% in distilled water and saved at 4°C. Fluoro-Ruby (FR, Molecular Probes, Eugene, OR, United States) and CTb (Recombinant), Alexa Fluor TM 488, and 647 Conjugate (CTb-488, CTb-647, Molecular Probes, Eugene, OR, United States) were dissolved at 10%, respectively, in normal saline and 1X phosphate-buffered saline (PBS) and saved at –20°C. All these tracers were kept on the ice during preparation and injection.
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