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Fast blue fb

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Fast Blue (FB) is a laboratory reagent used for staining and visualization purposes. It is a dye that can be used to detect the presence of specific biomolecules or cellular structures. The core function of Fast Blue is to provide a color contrast that allows for the identification and localization of target analytes in samples.

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Complete freund adjuvant (cfa), by Chondrex (1 mentions) Complete freund s adjuvant cfa, by Chondrex (1 mentions) Lister hooded rats, by Charles River Laboratories (1 mentions)

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Fast Blue (30 citations)

12 protocols using fast blue fb

1

Labeling Colonic Afferent Neurons

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Colonic afferent neurons were specifically labeled by injecting conventional neuronal tracing dye to the distal colon as described previously by us (Qiao and Grider, 2007 (link)). Briefly, the rat distal colon was exposed with a lower abdominal incision under anesthesia (2.5 % isoflurane) and in a sterile environment. Fast Blue (FB, 4 %, weight/volume; Polysciences, Inc. Warrington, PA) was injected into 10 sites (4 µL per site) in the muscular wall of the descending colon 5–7 cm away proximal to the external anal sphincter to label colonic afferent neurons innervating this area. Sterilized Hamilton syringe (10 µL size) was used for injection. Injections into the lumen, major blood vessels, or overlying fascial layers were avoided. The incision was closed with 4-0 sutures, and the rats were allowed to survive until the harvest of the tissues. FB injection was performed 4 days prior to TNBS instillation to ensure proper transport of the tracing dye to the DRG. These animals further underwent intracardiac perfusion and tissues were used for immunohistochemistry only.
Shen S., Al-Thumairy H.W., Hashmi F, & Qiao L.Y. (2017). Regulation of transient receptor potential cation channel subfamily V1 protein synthesis by the phosphoinositide 3-kinase/Akt pathway in colonic hypersensitivity. Experimental neurology, 295, 104-115.
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2

Anatomical Connectivity Mapping via Tracers

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Retrograde and anterograde tracers were injected in cortical sites to examine their anatomical connectivity (AC). Injections were in a subset of sites in M1, PMd, and PMv that had been investigated with ICMS+ISOI. Tracers were pressure injected using Hamilton syringes fitted with a beveled glass pipette. For each site, tracer was injected 700 μm and 1400 μm below the cortical surface. Four tracers were used: (1) Cholera toxin B subunit (CTB, SigmaMillipore). (2) Biotinylated dextran amine 1:1 mixture of 3,000 MW and 10,000 MW (BDA, ThermoFisher Scientific). (3) Dextran tetramethylrhodamine 3,000 MW, commonly known as fluoro ruby (FR, ThermoFisher Scientific). (4) Fast blue (FB, Polysciences). Total volumes were 0.4 μL for FB (2% in dH2O), 0.8 μL for FR (10% in dH2O), 0.6 μL for CTB (1% in dH2O), and 0.8–1.5 μL for BDA (10% in dH2O). After 12–13 days of tracer transport, a terminal experiment was conducted for additional ISOI recordings and microelectrode mapping. Also, microlesions were made (5 μA DC) to create salient landmarks for co-registering histological sections, ISOI maps, and microelectrode maps.
Card N.S, & Gharbawie O.A. (2022). Cortical connectivity is embedded in resting state at columnar resolution. Progress in neurobiology, 213, 102263.
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3

Intra-articular Injection of Retrograde Tracer and CFA

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Under anesthesia (100 mg/kg ketamine and 10 mg/kg xylazine, intraperitoneally), mice were injected intra-articularly through the patellar tendon into each knee with the retrograde tracer, fast blue (FB, 1.5 μL 2% in 0.9% saline, Polysciences) or into the left knee with 7.5 μL CFA (10 mg/mL, Chondrex). Vernier calipers were used to measure knee width (as before13 (link)), both before and 24 hours after CFA injection.
Chakrabarti S., Hore Z., Pattison L.A., Lalnunhlimi S., Bhebhe C.N., Callejo G., Bulmer D.C., Taams L.S., Denk F, & Smith E.S. (2020). Sensitization of knee-innervating sensory neurons by tumor necrosis factor-α-activated fibroblast-like synoviocytes: an in vitro, coculture model of inflammatory pain. Pain, 161(9), 2129-2141.
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4

Retrograde Tracing of BLA and VS

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Prior to avoidance conditioning, rats were anesthetized with isoflurane inhalant gas (5%) in an induction chamber and positioned in a stereotaxic frame. Isoflurane (1–2%) was delivered through a face mask for anesthesia maintenance. Rats were stereotaxically infused in the right hemisphere with retrograde tracers in BLA and VS (Paxinos and Watson 2007 ). Cholera Toxin B (CTB, 594 nm, Thermofisher) was infused in BLA (0.25 μL; − 2.76 mm AP; ± 4.8 mm ML; − 8.9 mm DV) and Fast Blue (FB, 420 nm, Polysciences) was infused in VS (0.2 μL; + 1.68 mm AP; ± 1.10 mm ML; − 7.2 mm DV). Tracers were infused at a rate of 0.025 (BLA) and 0.020 (VS) μL/min over 10 min, and the injectors were left in place for additional 10 min to allow the tracers to diffuse. Afterwards, rats recovered for 1 week prior to behavioral experiments.
Martínez-Rivera F.J., Bravo-Rivera C., Velázquez-Díaz C.D., Montesinos-Cartagena M, & Quirk G.J. (2018). Prefrontal circuits signaling active avoidance retrieval and extinction. Psychopharmacology, 236(1), 399-406.
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5

Retrograde Tracing of Gastrocnemius Motor Pool

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To identify the gastrocnemius (G) motor pool on both sides of the spinal cord, the G muscles were bilaterally injected with retrograde tracers 2–3 d before P10. Depending on survival times, we used a short-term tracer [cholera subunit b coupled to Alexa Fluor 555; CTb-555 (CTb), Thermo Fisher Scientific] or a long-term tracer [Fast Blue (FB), Polysciences]. Two 1 μl injections of 1% CTb-555 diluted in sterile saline or 2.5% FB diluted in sterile water were injected at different locations into the body of the muscle with glass micropipettes (Sigma-Aldrich). CTb-555-injected animals were analyzed 1 and 2 weeks postinjury, and FB-injected animals were used for the 60 d survival time point.
Arbat-Plana A., Bolívar S., Navarro X., Udina E, & Alvarez F.J. (2023). Massive Loss of Proprioceptive Ia Synapses in Rat Spinal Motoneurons after Nerve Crush Injuries in the Postnatal Period. eNeuro, 10(2), ENEURO.0436-22.2023.
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6

Labeling Bladder Afferent Neurons with Fast Blue

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Bladder afferent neurons were specifically labeled by conventional neuronal tracing dye Fast Blue (FB, 4 %, weight/volume; Polysciences, Inc. Warrington, PA) in live animals through surgery as described previously (Qiao and Grider, 2007 (link)). Briefly, the rat urinary bladder was exposed with a lower abdominal incision under anesthesia (2.5 % isoflurane). FB was injected into 10 sites (4 μL per site) in the muscular wall of the bladder to label bladder afferent neurons in the DRG. Sterilized Hamilton syringe (10 μL size) was used for injection. To prevent leakage and labeling of adjacent tissues, a cotton swab was used, close to the injection site, to wipe off any excess dye that might leak during needle withdrawal after each injection. We also avoided to inject dyes into the lumen, major blood vessels, or overlying fascial layers. The incision was closed with 4–0 sutures. The surgeries were done 7 days prior to harvest of tissues. We also checked the leakage of the dyes by examining the bladder and surrounding tissues after euthanasia of the rats. No contamination was found.
Xia C., Shen S., Hashmi F, & Qiao L.Y. (2015). Colitis-induced bladder afferent neuronal activation is regulated by BDNF through PLCγ pathway. Experimental neurology, 285(Pt B), 126-135.
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7

Mapping Sensory Neurons in Mice and Rats

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Fast Blue (FB; 2% in saline, Polysciences Gmbh, Germany) was injected into the wall of the distal colon of NaV1.9+/+ and NaV1.9−/− mice and male Sprague Dawley rats (150–250 g). Briefly, animals were anaesthetized with isoflurane then an approximate 1.5 cm laparotomy performed. Five injections of 0.2 μL FB per site, at a rate of 0.4 μL/min via a microinfusion pump, were made into the wall of the distal colon using a fine-pulled glass needle. The muscle layer was sutured and the skin secured with Michel clips. Postoperative analgesia (buprenorphine 0.05–0.1 mg/kg daily) and care (monitoring body weight and soft diet) was provided.
After 3 days for mice and 7 days for rats, animals were euthanized with sodium pentobarbital (200 mg/kg i.p.) and transcardially perfused with saline (0.9%) followed by paraformaldehyde (4% in 0.1 M phosphate buffer; pH 7.4). Dorsal root ganglia (DRG; NaV1.9+/+ and NaV1.9−/− mice, T13–L1; rats, L2) were removed and postfixed in 4% paraformaldehyde for 2 h and cryoprotected in 30% sucrose (w/v phosphate-buffered saline). Cryostat sections (10 μm) were collected sequentially across 10 slides per DRG.
Hockley J.R., Boundouki G., Cibert-Goton V., McGuire C., Yip P.K., Chan C., Tranter M., Wood J.N., Nassar M.A., Blackshaw L.A., Aziz Q., Michael G.J., Baker M.D., Winchester W.J., Knowles C.H, & Bulmer D.C. (2014). Multiple roles for NaV1.9 in the activation of visceral afferents by noxious inflammatory, mechanical, and human disease–derived stimuli. Pain, 155(10), 1962-1975.
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8

Retrograde Tracer Labeling and Knee Inflammation

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Under anesthesia (ketamine, 100 mg/kg and xylazine, 10 mg/kg, i.p.) a single injection of the retrograde tracer Fast Blue (FB; 1.5 μl 2% in 0.9% saline; Polysciences) was made intra-articularly through the patellar tendon into each knee to label knee-innervating neurons. For all experiments, anaesthetized mice were injected with 7.5 μl Complete Freund's adjuvant (CFA; 10 mg/ml; Chondrex) intra-articularly through the patellar tendon in the left knee seven days after administration of FB. Knee width was measured with Vernier's calipers before and 24-h after CFA injection.
Chakrabarti S., Pattison L.A., Singhal K., Hockley J.R., Callejo G, & Smith E.S. (2018). Acute inflammation sensitizes knee-innervating sensory neurons and decreases mouse digging behavior in a TRPV1-dependent manner. Neuropharmacology, 143, 49-62.
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9

Labeling Bladder Afferent Neurons in Spinal Cord Injury Mice

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A total 28 female C57BL/6N mice were used. At 4 weeks after SCI or sham operation, L6 DRGs were removed bilaterally in a separate group of SI (n = 8) and SCI (n = 5). To label bladder afferent neurons, under isoflurane anesthesia, animals received an injection of a total of 20-µL Fast Blue (FB) (1.5% w/v in water, Polysciences Inc., Warrington, PA, USA) at four sites in the bladder wall (5-µl per site) using a 31 -gauge Hamilton syringe into the bladder wall, 1 week prior to tissue removal.
Shimizu N., Doyal M.F., Goins W.F., Kadekawa K., Wada N., Kanai A.J., de Groat W.C., Hirayama A., Uemura H., Glorioso J.C, & Yoshimura N. (2017). Morphological Changes in Different Populations of Bladder Afferent Neurons Detected by Herpes Simplex Virus (HSV) Vectors with Cell Type-Specific Promoters in Mice with Spinal Cord Injury. Neuroscience, 364, 190-201.
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10

Retrograde Tracer Injections in Rat RSC

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Twenty-three male Lister hooded rats (Charles River, UK; Envigo, UK) weighing 291-614 g at the time of surgery were used for the retrograde tracer experiments. Four retrograde tracers were employed for a total of 29 injections: (i) cholera toxin subunit B (CTB; Polysciences Inc, Eppelheim, Germany), (ii) fast blue (FB; Polysciences Inc, Eppelheim, Germany), (iii) biotinylated dextran amine tracer (BDA; 3 kD version; Life Technologies Ltd, Paisley, UK), and (iv) fluorogold (FG; Fluorochrome Inc., Denver, CO, USA). The CTB was used either unconjugated or conjugated with Alexa Fluor 594 (Ctb-AF594; Molecular Probes, Invitrogen Inc.) or with Alexa Fluor 488 (Ctb-AF488; Molecular Probes, Invitrogen Inc.). The single BDA case was also included in a previous study (Mathiasen et al., 2017) , which provides full methodological procedures for this tracer (same study included FB cases, 64#3; 172#27; 172#28). Six animals had dual injections while the others had single injections placed in either gRSC or dRSC.
Lomi E., Mathiasen M.L., Cheng H.Y., Zhang N., Aggleton J.P., Mitchell A.S, & Jeffery K.J. (2021). Evidence for two distinct thalamocortical circuits in retrosplenial cortex. Neurobiology of learning and memory, 185.
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