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Ctb alexa 488

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The CTB-Alexa-488 is a fluorescently labeled cholera toxin B subunit. It is used as a marker for visualizing and tracing cellular uptake and trafficking processes.

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Lab products found in correlation

Lsm 510 microscope, by Zeiss (2 mentions) Gaasp photomultiplier tube, by Hamamatsu Photonics (1 mentions) Ff01 625 90 30 d, by IDEX Corporation (1 mentions) 27 g needle, by Terumo (1 mentions) Ctb alexa 555, by Thermo Fisher Scientific (1 mentions) Alexa 647 ctb, by Thermo Fisher Scientific (1 mentions) Ambroxol, by Merck Group (1 mentions) 10 μl syringe, by Hamilton Company (1 mentions)

Close spelling variants of this product

Alexa 488-conjugated CTB CTB-488 Alexa Fluor conjugate Alexa Fluor 488‐conjugated CTB Alexa Fluor 488-conjugated cholera toxin-β (CTB, CTB-488 Alexa 488

11 protocols using ctb alexa 488

1

Bilateral CTB Injection Protocol

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Bilateral CTB injection was performed as previously described (Riccomagno et al., 2014 (link)). Briefly, the adult animals were anesthetized using isophorone and then injected with 2 µL CTB-Alexa-555 or CTB-Alexa-488 (Life Technologies, 1mg/mL) bilaterally into the vitreous of each eye.
Sun L.O., Brady C.M., Cahill H., Al-Khindi T., Sakuta H., Dhande O.S., Noda M., Huberman A.D., Nathans J, & Kolodkin A.L. (2015). Functional Assembly of Accessory Optic System Circuitry Critical for Compensatory Eye Movements. Neuron, 86(4), 971-984.
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2

Retrograde Tracing of S2 Neurons

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Two of the three mice used for awake S1 somata imaging (Fig. 4) were subsequently injected with a retrograde tracer, CTB-Alexa488 (5 μg μl−1 in PBS, Life Technologies) in S2, as localized by intrinsic signal imaging. A small hole was drilled through the glass cranial window. One-hundred nanoliters of CTB-Alexa488 was injected (depth, 300–500 μm; rate, ~1 nl s−1) through the hole via a glass pipette (30–50 μm). The injection site was then sealed with dental cement. Seven days after the injection, labeled cells were examined under the two-photon microscope. To localize co-labeling, jRGECO1a-labeled cells and CTB-Alexa488-labeled cells were excited at 1,040 nm or 960 nm, (40–60 mW at specimen), respectively. Fluorescence emission was separated using a 568 nm dichroic (FF568-Di01-35.5×50.2, Semrock), passed through red (FF01-625/90-30-D, Semrock) and green (ET525/70m-2p, Chroma) channel filters before detection with two GaAsP photomultiplier tubes (Hamamatsu). We observed little fluorescence from jRGECO1a in the green channel, allowing us to use this channel to identify neurons labeled with CTB-Alexa488. The field of view was 273 μm × 298 μm (440 × 512 pixels; pixel size, 0.62 μm × 0.58 μm).
Kwon S.E., Tsytsarev V., Erzurumlu R.S, & O’Connor D.H. (2017). Organization of orientation-specific whisker deflection responses in layer 2/3 of mouse somatosensory cortex. Neuroscience, 368, 46-56.
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3

Retrograde Labeling of Cervical Motor Neurons

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Retrograde labeling of cervical MNs with cholera toxin subunit B-conjugated Alexa Fluor 488 (CTB-Alexa 488, Life Technologies) was carried out according to a standard procedure described previously12 (link),29 (link)–34 (link). On P6-P7, mice were anesthetized using 2.8% isoflurane. To retrogradely label forearm MN pools in the cervical spinal cord, we used a 10-µl Hamilton syringe fitted with a 27 G needle (Terumo) to inject forearm muscles with CTB-Alexa 488 diluted to 1 mg/ml in PBS. CTB-Alexa 488 is taken up from the axons and transported to and accumulated in the MN somata. Restriction of CTB-labeled neurons to the MNs is based on the fact that MNs are only neurons in the spinal cord extending the axons to the skeletal muscles.
Murabe N., Mori T., Fukuda S., Isoo N., Ohno T., Mizukami H., Ozawa K., Yoshimura Y, & Sakurai M. (2018). Higher primate-like direct corticomotoneuronal connections are transiently formed in a juvenile subprimate mammal. Scientific Reports, 8, 16536.
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4

Unilateral AAV Injection and Regenerative Labeling

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Female or male mice (5–7 weeks old) received unilateral AAV injection. For each intravitreal injection, a glass micropipette was inserted into the peripheral retina, just behind the ora serrata, and was deliberately angled to avoid damage to the lens. To anterogradely label regenerating RGC axons, animals received intravitreal injection of 2 μl of cholera toxin β subunit (CTB)-Alexa 555 or CTB-Alexa 488 (2 μg/μl, Invitrogen, Waltham, MA, USA) with a Hamilton syringe (Hamilton, Reno, NE, USA) at 2–5 days before euthanasia.
Luo X., Ribeiro M., Bray E.R., Lee D.H., Yungher B.J., Mehta S.T., Thakor K.A., Diaz F., Lee J.K., Moraes C.T., Bixby J.L., Lemmon V.P, & Park K.K. (2016). Enhanced Transcriptional Activity and Mitochondrial Localization of STAT3 Co-induce Axon Regrowth in Adult Central Nervous System. Cell reports, 15(2), 398-410.
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5

Mapping Collateralized MS Neuron Projections

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We exploited three methods to investigate the collateralization of LHb- and POA-projecting MS neurons: 1) AAVretro-GFP and AAVretro-Cre (Tervo et al., 2016 (link)) were injected into LHb and POA respectively of an Ai14 mouse (Figure 6D), which could retrogradely label the populations projecting to the corresponding targets; 2) CTb-Alexa647 and CTb-Alexa488 (Invitrogen) were injected into LHb and POA respectively of a wild-type mouse (Figure S7A); 3) AAV-EF1a-DIO-flp was injected into POA of a VGlutT2-Cre mouse, which would be retrogradely transported (Zingg et al., 2017 (link)) to MS and allow expression of flippase (FLP) in glutamatergic MS neurons, and this was followed by injection of AAVDJ-EF1a-fDIO-YFP (UNC Vector Core) into MS, allowing FLP-dependent expression of YFP (Figure S7B).
Zhang G.W., Shen L., Zhong W., Xiong Y., Zhang L.I, & Tao H.W. (2018). Transforming Sensory Cues into Aversive Emotion via Septal-Habenular Pathway. Neuron, 99(5), 1016-1028.e5.
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6

Ambroxol Promotes Optic Nerve Regeneration

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All experimental procedures were performed in compliance with animal protocols approved by Institutional Animal Care and Use Committee (IACUC) at Boston Children’s Hospital. ONs from 6-week-old C57BL/6 mice were crushed as described previously (Park et al., 2008 (link)) just after intravitreal injection of 1 µl ambroxol (Sigma, 25 mg/ml in 5% Tween 80%-5% Polyethylen glycol 400 in water) or vehicle (5% Tween 80%-5% PEG 400 in water). A second intravitreal injection of ambroxol or vehicle was performed 7 days post-ON crush. Daily mice received 120 µl (25 mg/ml) ambroxol or vehicle intraperitoneally, i.e., from the day after the ON crush to the day prior to termination. For PTEN−/− animal experiments, P21 PTEN floxed mice received AAV2-Cre intraorbital injection. Then 2 weeks later we performed ON crush and the first ambroxol injection within the eye. We injected (intraperitoneally) ambroxol at 300 mg/kg for the first 5 days after the crush and then we injected 150 mg/kg. Mice were injected twice a day every day. Regenerating axons were traced by intravitreal injection of 1 µl CTB-Alexa-488 (1 µg/µl in PBS, Invitrogen) 2 days before termination.
Chandran V., Coppola G., Nawabi H., Omura T., Versano R., Huebner E.A., Zhang A., Costigan M., Yekkirala A., Barrett L., Blesch A., Michaelevski I., Davis-Turak J., Gao F., Langfelder P., Horvath S., He Z., Benowitz L., Fainzilber M., Tuszynski M., Woolf C.J, & Geschwind D.H. (2016). A Systems-Level Analysis of the Peripheral Nerve Intrinsic Axonal Growth Program. Neuron, 89(5), 956-970.
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7

Photobleaching of Alexa 488-CTB

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Cells were incubated with Alexa 488-CTB (Invitrogen) in the dark for 30 min on ice. Cells were briefly rinsed with ice-cold media and placed on ice in the dark until use. Imaging and photobleaching were conducted on a Zeiss LSM510 microscope (Carl Zeiss) equipped with an Ar 488 nm laser. 60 iterations at 100% of 488 nm excitation was used to photobleach a small portion (31 px diameter) of the membrane. Adjacent non photobleached membrane areas of the same size were used as a control for sequential bleaching as previously described (Tecedor et al., 2013 (link)).
Schultz M.L., Tecedor L., Lysenko E., Ramachandran S., Stein C.S, & Davidson B.L. (2018). Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo. Neurobiology of disease, 115, 182-193.
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8

Photobleaching of Alexa 488-CTB

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Cells were incubated with Alexa 488-CTB (Invitrogen) in the dark for 30 min on ice. Cells were briefly rinsed with ice-cold media and placed on ice in the dark until use. Imaging and photobleaching were conducted on a Zeiss LSM510 microscope (Carl Zeiss) equipped with an Ar 488 nm laser. 60 iterations at 100% of 488 nm excitation was used to photobleach a small portion (31 px diameter) of the membrane. Adjacent non photobleached membrane areas of the same size were used as a control for sequential bleaching as previously described (Tecedor et al., 2013 (link)).
Schultz M.L., Tecedor L., Lysenko E., Ramachandran S., Stein C.S, & Davidson B.L. (2018). Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo. Neurobiology of disease, 115, 182-193.
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9

Tracing ArcN NPY/AgRP Neuron Projections

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To determine if NPY that binds to DMH Y1R originates from neurons in
the ArcN, we identified ArcN BAT pre-sympathetic neurons by injecting the
retrogradely transported indicator Cholera toxin B (CtB; Alexa-488-CtB
1mg/ml, 30 nL, Life Technologies, Invitrogen™, Carlsbad,
CA) into the RPa (7 (link)). Using the
Cre-dependent synaptically-targeted AAV-Syn-mCherry injected bilaterally
into the ArcN (coordinates: 1.0 to 1.6 mm caudal to bregma, 0.3 mm lateral
to midline, 5.8 mm ventral to dura) of AgRP-ires-Cre mice,
confocal microscopy (LSM 880indimo AxioObserver with 63x/1.4 Oil immersion
objective imaging 16–5 μm optical sections), and ZenBlue
software, we tested if DMH CtB labeled neurons are closely associated with
ArcN NPY/AgRP neuronal terminals.
Shi Z., Bonillas A.C., Wong J., Padilla S.L, & Brooks V.L. (2021). Neuropeptide Y suppresses thermogenic and cardiovascular sympathetic nerve activity via Y1 receptors in the paraventricular nucleus and dorsomedial hypothalamus. Journal of neuroendocrinology, 33(8), e13006.
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10

Retrograde Tracing of Dental Pulp Innervation

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The surgery was performed under anesthesia with 2% isoflurane and mice were aligned in a stereotactic frame (KOPF instruments, Tujunga, CA). Briefly, hair over the cheek was clipped, and the skin was aseptically prepared using alternating betadine and alcohol scrubs. Sagittal incisions were made on the skin and masseter muscle to expose the incisor. Two full-thickness defects of 0.3–0.4 mm diameter and 2.5–3.0 mm apart were created on the bone overlying the incisor mesenchyme using a microsurgical drill and a trephine drill bit. A 10 μl syringe (Hamilton Company, Reno, NV) coupled to a glass capillary pulled pipette was inserted into one of the bone defects and calking material was applied around the glass pipette to form an airtight seal. 500 nl 2% CTB-Alexa488 (Molecular Probes: C-34775) was injected into the incisor mesenchyme over a period of 2 min. The glass pipette was kept in the bone defect for 15 min to allow the retrograde tracer to be fully absorbed by the dental pulp. The masseter muscle and cheek skin were then sutured closed. The mice were then subcutaneously injected with Buprenorphine SR (1 mg/kg) and allowed to recover on a heating pad until fully awake. Trigeminal ganglia were harvested up to 72 h after injection.
Pei F., Ma L., Jing J., Feng J., Yuan Y., Guo T., Han X., Ho T.V., Lei J., He J., Zhang M., Chen J.F, & Chai Y. (2023). Sensory nerve niche regulates mesenchymal stem cell homeostasis via FGF/mTOR/autophagy axis. Nature Communications, 14, 344.
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