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-range transport of biocytin has been used to trace neuronal tracts under in vivo or in vitro conditions (Kobbert et al., 2000, Thomson and Armstrong, 2011). Uptake can be facilitated by co-injection of NMDA or KCL (Chang et al., 2000, Jiang et al., 1993, Tarras-Wahlberg and Rekling, 2009, Zheng et al., 1998), but it is not entirely clear how extracellular deposited biocytin is taken up, nor have the mechanisms responsible for active transport in tracts been identified. However, the molecule appears to have affinity for some component in the transport machinery, and we have utilized this principle here to demonstrate that the fluorophore TMR coupled by a long spacer to biocytin acts as a fast retrograde tracer that can be used to label live neurons over long distances in a matter of hours.[1]
TMR biocytin had the highest transport velocity in spinal cord tracts (5.4 mm/h) of the six compounds we tested. Unconjugated TMR was not taken up into fibers, suggesting that the biocytin moiety is critical for uptake and fiber transport. Several other biotin conjugated fluorescent compounds were taken up and transported, notably Atto 488 Biotin, which was transported with a velocity close to that of TMR biocytin. Biocytin-TMR and Atto 488 Biotin have red and green emission spectra respectively and consequently can be used in dual labeling experiments. However, Atto 550 Biotin, showed very little uptake and slow transport velocities. We hypothesize that at least two chemical properties may contribute to the uptake and transport properties of the tested conjugated compounds. First, recent data show that the fluorophore moieties of the tested compounds have very different interactions with lipid bilayers, e.g. the fluorophore of the slowly transported Atto 550 Biotin interacts strongly with lipid bilayers (MIF: 33), whereas the fluorophore of Biocytin-TMR interacts weakly (MIF: 0.35,(Hughes et al., 2014). This relationship suggests that compounds with a strong lipid bilayer interaction may get trapped in lipids and cannot be recruited by the axonal transport machinery. Second, the spacer length between the fluorophore and the biotin/biocytin moiety differs among the tested compounds, i.e. TMR biocytin and Atto 488 Biotin have 20- and 18-carbon–nitrogen aliphatic spacers (Fig. 4), whereas the slower transported Atto 565 Biotin has a 13-carbon-nitrogen long aliphatic spacer. Thus, spacer length may also be a contributing factor to the transport properties of the compounds by allowing more or less space for molecular interactions necessary for transport. We have opted to perform experiments in young postnatal animals to be able to maintain large pieces of nervous tissue viable under in vitro conditions. At the oldest age (P15.5) myelination has begun, and consequently we expect TMR biocytin will also work in older adult animals, but it remains to be shown in separate experiments.[1]
Several properties make TMR biocytin a very attractive neuronal tracer. The fluorophore emits light in the red end of the visual spectrum, which is an advantage since the longer emission wavelength means less scatter in the tissue thereby improving visualization of deep structures. Thus, using confocal microscopy on methyl salicylate cleared tissue we visualized Biocytin-TMR labeled fiber tracts to a depth of more than 0.5 mm (Fig. 7). Some of the best calcium sensors based on synthetic small-molecule organic dyes, e.g. Fluo-4, Fluo-8, Oregon Green, and Calcium Green have emission spectra (Em: 506–531 nm) that can be effectively separated from TMR biocytin emission (Em: 581 nm). Recently improved genetically encoded calcium sensors such as the GCaMP family also have shorter wavelength emission peaks (Em: ∼512 nm (Badura et al., 2014). Thus, neurons can be dual labeled with a calcium sensor and TMR biocytin for concurrent calcium imaging and somadendritic visualization. The duration of spontaneous calcium transients in Fluo-8 AM/TRM Biocytin labeled IO neurons was not affected by TMR biocytin, and Fluo-8 AM/TRM Biocytin labeled organotypical slice cultures showed spontaneous oscillations in dual labeled neurons. This we interpret as sign of good neuronal viability for the illumination parameters used here, i.e. low phototoxicity is expected in the use of TMR biocytin. The high transport velocity of TMR biocytin makes it possible to perform these types of experiments over the course of a few hours, as demonstrated here by imaging of oscillating commissural neurons in organotypical slice cultures containing respiratory neurons (Fig. 5). Similar experiments using Calcium Green-1 AM injections in the midline of acutely prepared brainstem slices containing oscillating commissural neurons required overnight incubation (Koshiya and Smith, 1999). Retrogradely transported TMR biocytin also Biocytin also passes into dendrites (Fig. 1, Fig. 7), which opens for experiments involving targeted dendritic recordings and dual labeling using AM dyes, which in some cases also label proximal dendrites (Del Negro et al., 2011). Juxtacellular electroporation of Biocytin-TMR leads to strong somadendritic labeling, and anterograde transport into local axonal arborisations with visible synaptic profiles within 10 min (Fig. 7). This anterograde transport could conceivable be used in uncovering local circuit motifs, targeted presynaptic experiments, and combined morphological and electrophysiological characterizations. TMR biocytin was readily taken up by cranial nerve roots (Fig. 7), and thus shows promise as an effective alternative to carbocyanine dyes such as DiO and DiI often used in developmental neurobiology to label defined cranial and spinal compartments. Finally, the presence of a lysine moiety in TMR biocytin makes it fixable with paraformaldehyde and glutaraldehyde, which aid morphological visualization in fixed and cleared tissue.[1]
Using a similar rationale (Mishra et al., 2011) developed a biocytin-derived MRI contrast agent and demonstrated long-range retrograde transport of this functionalized tracer. It is not difficult to imagine how further development of the principle of a biocytin/biotin moiety coupled to a functional fluorophore may lead to reporter molecules that can be transported at high velocity in neuronal tracts and report on e.g. calcium, sodium, or chloride concentrations in synapses, dendrites, somas, or axons. However, the chemical properties of the functional fluorophore might be critical for successful transport.[1]

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Previous studies suggested that ECS may cause a breakdown in the blood brain barrier (BBB) (Ito et al., 2017), raising the possibility that the change in CD11b + cell expression signature might represent an influx of blood monocytes. We therefore examined the effect of ECS on the BBB by use of Biocytin-TMR tracer. First, we confirmed that systemically administered Biocytin-TMR penetrates the brain parenchyma in acute EAE, a model of neuroinflammation (Fig. 4A-D). Injection of Biocytin-TMR at one-hour post ECS and 24 h post three daily ECS sessions in naïve mice showed an intact BBB (Fig. 4E-H). We also examined whether the differentially expressed genes correspond to genes that were reported as representing infiltrating monocytes / macrophages. Only two out of 98 upregulated genes matched genes that defined infiltrating monocytes in acute EAE (Yamasaki et al., 2014) (Fig. 4I), and only three out of 98 genes matched those that defined infiltrating monocytes / macrophages in virus-induced neuroinflammation (DePaula-Silva et al., 2019). Although markers of infiltrating monocytes were described in conditions of neuroinflammation rather than in healthy CNS, these paucity of genes representing infiltrating monocytes suggest that ECS induced a phenotypic shift in resident CNS derived CD11b+ cells, rather than induced massive influx of peripheral monocytes. Single cell analysis may further reveal whether there are changes in small subsets of CNS monocular cells.[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.)