Retrograde tracing was performed on axons of DRG neurons using a previously described protocol (Liu et al., 2009). Briefly, 5 days before the animals were sacrificed, the cats were given general anesthesia and the bilateral lumbosacral trunks were isolated. Cholera toxin B subunit conjugated to horseradish peroxidase (CB-HRP) 15 µL (30%; Sigma-Aldrich, St. Louis, MO, USA) was injected into the bilateral lumbosacral trunks for retrograde labeling of DRG neurons. Staining was performed using tetramethylbenzidine (TMB). Briefly, the L6 DRG and spinal cord segment were fixed in 4% paraformaldehyde and stained with TMB (1%; Sigma-Aldrich). PBS instead of CB-HRP was used for the negative control. The area of the spinal dorsal horn was measured. CB-HRP-positive nerve fibers were counted under a microscope (X51, Olympus) to calculate their density in the dorsal horn. The final measurements were the average of the five selected sections from each cat.
Cb hrp
Manufactured by Merck Group
Sourced in United States
CB-HRP is a horseradish peroxidase (HRP) conjugate designed for use in various immunoassay applications. HRP is an enzyme widely used as a label in immunodetection techniques due to its ability to catalyze color-producing reactions. The CB-HRP product provides a consistent and reliable HRP conjugate for researchers and scientists.
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5 protocols using cb hrp
1
Retrograde Tracing of DRG Neurons
2
Tract-Tracing of CSF-Contacting Nucleus
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Monkeys were anesthetized with ketamine (10 mg/kg, i.m.), and the head was fixed on a stereotaxic instrument. 50 μl 30% CB-HRP (Sigma, United States), a specific tracer of the CSF-contacting nucleus through the ventricular system, was injected into the LV according to the stereotaxic coordinates provided by Paxinos et al. (2000) . The successful injection was confirmed by extracting the CSF using a microsyringe. After 48 h, the monkeys were perfused.
3
CB-HRP Tracing of CSF-Contacting Nucleus
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Rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.), and heads were fixed on the stereotaxic instrument (Stoelting, United States). Then, 3 μl 30% CB-HRP (Sigma, United States), a specific tracer of the CSF-contacting nucleus by way of the ventricular system, was injected into the LV according to the stereotaxic coordinates provided by Paxinos and Watson (2007) . After 48 h, rats were perfused and sacrificed.
4
Retrograde Tracing of Rat Rectum Innervation
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With the rats (Group C, n = 10) lying supine under anesthesia, a median incision of the hypogastrium was made to expose the anal canal. CB-HRP (10 to 15 μl, 3 μg/μl) (Sigma, St Louise, MO, USA) was slowly injected into the anal canal wall using a micro-syringe into three sites equally distant from one to another. The needle was retained for 15 min and medical adhesive was applied to seal off the needle holes. The rats were kept alive for 48 h. Then, the rats were perfused transcardially with phosphate buffered saline (PBS), followed by buffered 4% paraformaldehyde. Spinal cords and bilateral dorsal root ganglia from T12 to S4 were removed and placed in PBS containing 20% sucrose at 4 °C for 24 hours. Forty-μm sections were obtained using a cryostat-microtome. HRP-positive neurons were shown using tetramethylbenzidine-sodium tungstate (TMB-ST) and observed using an Olympus BH-2 microscope equipped with brightfield and darkfield optics (Olympus, Tokyo, Japan). The proportions of CB-HRP-positive neurons in different segmental dorsal root ganglia were calculated using the Leica FW4000 image analysis system using one slide per animal.
CB-HRP positive neurons and afferent fibers of rat rectum (Group D, n = 10) from different spinal segments were analyzed using the same methodology as for the anal canal.
CB-HRP positive neurons and afferent fibers of rat rectum (Group D, n = 10) from different spinal segments were analyzed using the same methodology as for the anal canal.
5
Tracing Cerebrospinal Fluid Pathways
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Monkeys were anaesthetised with ketamine (10 mg/kg, i.m.), and the head was xed on a stereotaxic instrument. 50 µl 30% CB-HRP (Sigma, United States), a speci c tracer of the CSF-contacting nucleus through the ventricular system, was injected into the lateral ventricle according to the stereotaxic coordinates provided by Paxinos [9] . The successful injection was con rmed by extracting the CSF using a microsyringe. After 48 h, monkeys were perfused and sacri ced.
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