Fluorescent Dye

The blood brain barrier (BBB) permeability alterations can be investigated using TMR Biocytin[2]. Guidelines (This protocol is merely meant to serve as a guide; it should be adjusted to meet your unique requirements. The following is our suggested protocol)[2]. 1. For each mouse, dilute 1 milligram of TMR Biocytin in 100 μL of PBS, then inject the mixture into the tail vein. 2. Anesthetize and perfuse the animals half an hour after the injection. 3. Take off the spinal cords, then create serial 10 μm longitudinal sections that are deep frozen. 4. Use DAPI to stain nuclear counterstain. 5. Using the same laser intensity, exposure periods, and magnification for each cohort, capture photos of whole sections with a ×10 objective and ×10 eyepiece. 6. Liver samples from both tracer-injected and non-injected animals were utilized to set the aforementioned values.

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The initial fiber transport velocity of TMR biocytin is high-5.4mm/h. TMR biocytin can be used in conjunction with AM calcium dyes to label neuronal somas from distances of several millimetres, and record calcium transients during the course of a few hours. Juxtacellular application of TMR biocytin leads to fast anterograde transport with labeling of local synapses within 10min. TMR biocytin is fixable, stable during methyl salicylate clearing, and can be visualized deep in nervous tissue. Comparison with existing methods: Retrograde labeling with TMR biocytin enables long-range neuronal visualization and concurrent calcium imaging after only a few hours, which is substantially faster than other fluorescence-based tracers. The green emitting Atto 488 biotin is also taken up and transported retrogradely, but it is not compatible with standard green emitting calcium dyes. Conclusions: TMR biocytin is an attractive neuronal tracer. It labels neurons fast over long distances, and it can be used in conjunction with calcium dyes to report on neuronal activity in retrogradely labeled live neurons.[1]

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Brain stimulation by electroconvulsive therapy is effective in neuropsychiatric disorders by unknown mechanisms. Microglial toxicity plays key role in neuropsychiatric, neuroinflammatory and degenerative diseases. We examined the mechanism by which electroconvulsive seizures (ECS) regulates microglial phenotype and response to stimuli. Microglial responses were examined by morphological analysis, Iba1 and cytokine expression. ECS did not affect resting microglial phenotype or morphology but regulated their activation by Lipopolysaccharide stimulation. Microglia were isolated after ECS or sham sessions in naïve mice for transcriptome analysis. RNA sequencing identified 141 differentially expressed genes. ECS modulated multiple immune-associated gene families and attenuated neurotoxicity-associated gene expression. Blood brain barrier was examined by injecting Biocytin-TMR tracer. There was no breakdown of the BBB, nor increase in gene-signature of peripheral monocytes, suggesting that ECS effect is mainly on resident microglia. Unbiased analysis of regulatory sequences identified the induction of microglial retinoic acid receptor α (RARα) gene expression and a putative common RARα-binding motif in multiple ECS-upregulated genes. The effects of AM580, a selective RARα agonist on microglial response to LPS was examined in vitro. AM580 prevented LPS-induced cytokine expression and reactive oxygen species production. Chronic murine experimental autoimmune encephalomyelitis (EAE) was utilized to confirm the role RARα signaling as mediator of ECS-induced transcriptional pathway in regulating microglial toxicity. Continuous intracerebroventricular delivery of AM580 attenuated effectively EAE severity. In conclusion, ECS regulates CNS innate immune system responses by activating microglial retinoic acid receptor α pathway, signifying a novel therapeutic approach for chronic neuroinflammatory, neuropsychiatric and neurodegenerative diseases [2].

Optical recordings commenced 15–150 min after the preparation was placed in the recording chamber mounted on an Olympus BX51 microscope (upright, modified to be fixed stage, mounted on an XY platform). The recording chamber had a volume of 2 ml, a temperature of 29.5° C and was constantly superfused at a rate of 2 ml/min with preheated oxygenated (95% O2, 5% CO2) ACSF, which contained (in mM): 129 NaCl, 3 KCl, 5 KH2PO4, 25 NaHCO3, 30 d-(+)-glucose, 0.4 MgSO4 and 0.7 CaCl2. For detection of fluorescence signals, a metal halide light source PhotoFluor II or a LED light source, M470L2 (Thorlabs, New Jersey, USA) was coupled to a stereo microscope Olympus BX51 (upright, modified to be fixed stage, mounted on an XY platform) via a liquid light guide and appropriate optical filters (in nm): Biocytin-TMR, Atto 565 Biotin, Atto 565, Atto 550 Biotin, TMR; Olympus U-MWIG2, Excitation:BP520-550, DM565, Emission: BA580IF, and Atto 488 Biotin, Fluo-8 AM; Modified Olympus U-MWIB, Excitation:BP457-487, DM505, Emission: 515–550. Images were captured by an sCMOS camera controlled by the SOLIS software. Imaging was done using ×10, ×20, ×40 and ×63 water immersion objectives, at 1–10 frames/s, in image sessions lasting 1–20 s. Some Biocytin-TMR injected preparations were fixed overnight in 4% paraformaldehyde in 0.1 M Sorensen Phosphate buffer, dehydrated in increasing ethanol (20%, 30%, 50% 70%, 95%, 100%—10 min each), cleared in 100% methyl salicylate, and imaged using a Zeiss LSM 710 confocal microscope (Excitation: 561 nm, Emission: 623 nm). [1]

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Brain blood barrier permeability [2]
Four Biozzi ABH mice groups (3 mice per group) were evaluated; Naïve mice (negative control), EAE mice at peak of the first relapse (day 17 post-immunization, positive control), 1 h after a single ECS treatment and 24 h following 3 consecutive daily ECS treatment sessions. 1 mg of 5-(and 6)-tetramethylrhodamine biocytin (Biocytin-TMR) diluted in 100ul PBS was injected per mouse into the tail vein. Penetration into the blood circulation was indicated by pink colorization of the ears within seconds. 30 min after injection animals were anesthetized and perfused, spinal cords were removed, deep frozen and serial 10 μm longitudinal sections were prepared as described above. Nuclear counterstain was performed using DAPI (Vector Laboratories). Images of whole sections were obtained (×10 power of objective ×10 power of eyepiece) using identical laser intensity, exposure times and magnification in all cohorts. To set these parameters, livers from tracer injected mice and non-injected mice were used.

[1]. Fast neuronal labeling in live tissue using a biocytin conjugated fluorescent probe. J Neurosci Methods. 2015 Sep 30;253:101-9.

[2]. Electric neurostimulation regulates microglial activation via retinoic acid receptor α signaling. Brain Behav Immun. 2021 Aug;96:40-53.

Long-distance transport of biotin has been used to track nerve tracts in vivo or in vitro (Kobbert et al., 2000; Thomson and Armstrong, 2011). Co-injection of NMDA or KCl can promote biotin uptake (Chang et al., 2000; Jiang et al., 1993; Tarras-Wahlberg and Rekling, 2009; Zheng et al., 1998), but how extracellularly deposited biotin is taken up is not fully understood, and the mechanism responsible for active transport within nerve tracts is also unclear. However, the molecule appears to have an affinity for certain components of the transport mechanism, and we used this principle to demonstrate that the fluorophore TMR, coupled with biotin via a long septal arm, can serve as a rapid retrograde tracer to label long-distance live neurons within hours. [1]
Of the six compounds we tested, TMR biotin had the highest transport rate in the spinal tract (5.4 mm/h). Uncoupled TMR could not be absorbed by fibers, indicating that the biotin moiety is crucial for absorption and fiber transport. Several other biotin-conjugated fluorescent compounds were also absorbed and transported, notably Atto 488 biotin, which transported at rates close to those of TMR biotin. Biotin-TMR and Atto 488 biotin exhibit red and green emission spectra, respectively, and can therefore be used for double-labeling experiments. However, Atto 550 biotin showed extremely low absorption and slow transport. We hypothesize that at least two chemical properties may influence the absorption and transport characteristics of the tested conjugated compounds. First, recent data show significant differences in the interaction between the fluorophores of the tested compounds and the lipid bilayer. For example, the slower-transporting Atto 550 biotin exhibited a stronger interaction with the lipid bilayer (MIF: 33), while the fluorophore of Biotin-TMR showed a weaker interaction (MIF: 0.35, Hughes et al., 2014). This relationship suggests that compounds that interact strongly with the lipid bilayer may be captured by lipids and thus cannot be recruited by axonal transport mechanisms. Secondly, the length of the spacer between the fluorophore and the biotin/cytokinin moiety in the test compounds also differs. For example, TMR cytokinin and Atto 488 biotin have aliphatic spacers with 20 and 18 carbon-nitrogen atoms, respectively (Fig. 4), while Atto 565 biotin, which has a slower transport rate, has an aliphatic spacer with 13 carbon-nitrogen atoms. Therefore, the length of the spacer may also be a factor affecting the transport properties of the compound, as it determines the size of the space for molecular interactions required for transport. We chose to conduct experiments on young, postnatal animals to maintain the activity of large blocks of neural tissue under in vitro conditions. In the oldest animals (P15.5), myelination has begun, so we expect TMR cytokinin to be effective in older animals as well, but this still needs to be verified in separate experiments. [1] Several properties of TMR cytokinin make it a very attractive neuronal tracer. The fluorophore emits light at the red end of the visible spectrum, which is an advantage because the longer emission wavelength means less scattering in the tissue, thus improving the imaging of deep structures. Therefore, we used confocal microscopy to observe tissue cleared with methyl salicylate and observed fiber bundles labeled with biocytin-TMR at depths exceeding 0.5 mm (Figure 7). Some synthetic, high-performance small-molecule organic dyes based on calcium sensors, such as Fluo-4, Fluo-8, Oregon Green, and Calcium Green, have emission spectra (Em: 506–531 nm) that can be effectively separated from the emission spectrum of TMR biocytin (Em: 581 nm). Recently improved gene-encoded calcium sensors, such as the GCaMP family, also have shorter wavelength emission peaks (Em: ~512 nm (Badura et al., 2014)). Therefore, neurons can be dual-labeled using calcium sensors and TMR biocytin, enabling simultaneous calcium imaging and visualization of cell body dendrites. In IO neurons labeled with Fluo-8 AM/TRM biocytin, the duration of spontaneous calcium transients was unaffected by TMR biocytin, and in organoid culture cultures labeled with Fluo-8 AM/TRM biocytin, the double-labeled neurons exhibited spontaneous oscillations. We believe this indicates good neuronal survival under the illumination parameters used in this paper, suggesting low phototoxicity is expected when using TMR biotin. The high transport rate of TMR biotin allows such experiments to be completed within hours, as shown in this paper, by imaging oscillating commissural neurons in organoid culture cultures containing respiratory neurons (Figure 5). Similar experiments, such as midline injection of calcium green-1 AM into acutely prepared brainstem sections containing oscillating commissural neurons, require overnight incubation (Koshiya and Smith, 1999). Retrograde transport of TMR biotin also allows entry into dendrites (Fig. 1, Fig. 7), enabling experiments involving targeted dendritic recording and dual labeling with AM dyes, which in some cases can also label proximal dendrites (Del Negro et al., 2011). Paracellular electroporation of biotin-TMR results in strong cell-somatic dendritic labeling and anterograde transport to local axons. Dendritic structures with clear synaptic contours can be observed within 10 minutes (Fig. 7). This anterograde transport can be used to reveal local circuit motifs, target presynaptic experiments, and provide combined morphological and electrophysiological characterization. TMR biotin is readily absorbed by cranial nerve roots (Fig. 7), thus showing promise as an effective alternative to commonly used carbocyanine dyes (such as DiO and DiI) in developmental neurobiology for labeling specific cranial nerve and spinal cord regions. Furthermore, the presence of lysine residues in TMR biotin allows for fixation with paraformaldehyde and glutaraldehyde, facilitating morphological visualization in fixed and cleared tissues. [1]
Based on a similar principle (Mishra et al., 2011), they developed a biocytin-derived MRI contrast agent and demonstrated long-range retrograde transport of this functionalized tracer. It is not hard to imagine what the prospects would be for further development of this principle. Biotin/biotin moieties are coupled with functional fluorophores to form reporter molecules that can be transported at high speeds in nerve bundles and report the concentrations of calcium, sodium, or chloride ions in synapses, dendrites, cell bodies, or axons. However, the chemical properties of the functional fluorophore may be crucial for successful transport. [1]

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Previous studies have shown that ECS may lead to disruption of the blood-brain barrier (BBB) (Ito et al., 2017), suggesting that changes in CD11b+ cell expression characteristics may represent an influx of blood monocytes. Therefore, we investigated the effects of ECS on the BBB using a Biocytin-TMR tracer. First, we demonstrated that systemically administered Biocytin-TMR could penetrate the brain parenchyma in acute experimental autoimmune encephalomyelitis (EAE), a model of neuroinflammation (Fig. 4A-D). In untreated mice, biocytin-TMR staining at 1 hour after electroconvulsive therapy (ECS) and 24 hours after three consecutive days of ECS treatment showed an intact blood-brain barrier (BBB) (Fig. 4E-H). We also examined whether differentially expressed genes corresponded to previously reported genes representing infiltrating monocytes/macrophages. Of the 98 upregulated genes, only 2 matched genes defining infiltrating monocytes in acute experimental autoimmune encephalomyelitis (EAE) (Yamasaki et al., 2014) (Fig. 4I), while only 3 matched genes defining infiltrating monocytes/macrophages in virus-induced neuroinflammation (DePaula-Silva et al., 2019). Although the markers of infiltrating monocytes are described in a neuroinflammatory state rather than in a healthy central nervous system (CNS), the small number of genes representing infiltrating monocytes suggests that the ECS induces a phenotypic shift in resident CNS-derived cells, rather than inducing a large number of peripheral monocytes to infiltrate. Single-cell analysis may further reveal changes in small subsets of monocytes in the CNS. [2]

C46H60N8O7S

869.1

868.43056

749247-49-2

165412506

Typically exists as solid at room temperature

1.1

6

10

20

12

1710

4

CN(C)C1=CC2=C(C=C1)C(=C3C=CC(=[N+](C)C)C=C3O2)C4=C(C=C(C=C4)C(=O)NCCCCCNC(=O)[C@H](CCCCNC(=O)CCCC[C@H]5[C@@H]6[C@H](CS5)NC(=O)N6)N)C(=O)[O-]

RRJNRPHRYXQIAB-VSZNSILHSA-N

InChI=1S/C46H60N8O7S/c1-53(2)29-16-19-32-37(25-29)61-38-26-30(54(3)4)17-20-33(38)41(32)31-18-15-28(24-34(31)45(58)59)43(56)49-22-9-5-10-23-50-44(57)35(47)12-8-11-21-48-40(55)14-7-6-13-39-42-36(27-62-39)51-46(60)52-42/h15-20,24-26,35-36,39,42H,5-14,21-23,27,47H2,1-4H3,(H5-,48,49,50,51,52,55,56,57,58,59,60)/t35-,36-,39-,42-/m0/s1

5-[5-[[(2S)-6-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-2-aminohexanoyl]amino]pentylcarbamoyl]-2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]benzoate

TMR Biocytin; 749247-49-2;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)