Endogenous Metabolite

With a variety of avidin-conjugated markers, Biocytin may be seen at both the light and electron microscopic levels because of its strong affinity for avidin. The appearance of dendritic and axonal arborizations can be seen with the application of biocytin[1]. With a vast dendritic arbor and small population of ON cone bipolar cells, the Biocytin wide-field bipolar cell in rabbit retina does not make contact with every cone within its dendritic field. Cone types were identified using antibodies against blue cone opsin and red-green cone opsin, after which bipolar cells were identified by selective absorption of Biocytin and labeling of the cones with peanut agglutinin. The Biocytin-labeled cells avoid cones that do not stain for blue cone opsin and make selective contact with cones whose outer segments do. A homolog of the blue cone bipolar cells previously identified in the retinas of primates, mice, and ground squirrels, the biocytin wide-field bipolar cell is an ON blue cone bipolar cell found in the retina of rabbits[3].

One of the main characteristics of brains is their profuse connectivity at different spatial scales. Understanding brain function evidently first requires a comprehensive description of neuronal anatomical connections. Not surprisingly a large number of histological markers were developed over the years that can be used for tracing mono- or polysynaptic connections. Biocytin is a classical neuroanatomical tracer commonly used to map brain connectivity. However, the endogenous degradation of the molecule by the action of biotinidase enzymes precludes its applicability in long-term experiments and limits the quality and completeness of the rendered connections. With the aim to improve the stability of this classical tracer, two novel biocytin-derived compounds were designed and synthesized. Here we present their greatly improved stability in biological tissue along with retained capacity to function as neuronal tracers. The experiments, 24 and 96 h postinjection, demonstrated that the newly synthesized molecules yielded more detailed and complete information about brain networks than that obtained with conventional biocytin. Preliminary results suggest that the reported molecular designs can be further diversified for use as multimodal tracers in combined MRI and optical or electron microscopy experiments. [1]

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The connectivity and cytoarchitecture of telencephalic centers except dorsal and medial pallium were studied in the fire-bellied toad Bombina orientalis by anterograde and retrograde Biocytin labeling and intracellular Biocytin injection (total of 148 intracellularly labeled neurons or neuron clusters). Our findings suggest the following telencephalic divisions: (1) a central amygdala-bed nucleus of the stria terminalis in the caudal midventral telencephalon, connected to visceral-autonomic centers; (2) a vomeronasal amygdala in the caudolateral ventral telencephalon receiving input from the accessory olfactory bulb and projecting mainly to the preoptic region/hypothalamus; (3) an olfactory amygdala in the caudal pole of the telencephalon lateral to the vomeronasal amygdala receiving input from the main olfactory bulb and projecting to the hypothalamus; (4) a medial amygdala receiving input from the anterior dorsal thalamus and projecting to the medial pallium, septum, and hypothalamus; (5) a ventromedial column formed by a nucleus accumbens and a ventral pallidum projecting to the central amygdala, hypothalamus, and posterior tubercle; (6) a lateral column constituting the dorsal striatum proper rostrally and the dorsal pallidum caudally, and a ventrolateral column constituting the ventral striatum. We conclude that the caudal mediolateral complex consisting of the extended central, vomeronasal, and olfactory amygdala of anurans represents the ancestral condition of the amygdaloid complex. During the evolution of the mammalian telencephalon this complex was shifted medially and involuted. The mammalian basolateral amygdala apparently is an evolutionary new structure, but the medial portion of the amygdalar complex of anurans reveals similarities in input and output with this structure and may serve similar functions.[2]

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The Biocytin wide-field bipolar cell in rabbit retina has a broad axonal arbor in layer 5 of the inner plexiform layer and a wide dendritic arbor that does not contact all cones in its dendritic field. The purpose of our study was to identify the types of cones that this cell contacts. We identified the bipolar cells by selective uptake of Biocytin, labeled the cones with peanut agglutinin, and then used antibodies against blue cone opsin and red-green cone opsin to identify the individual cone types. The biocytin-labeled cells selectively contacted cones whose outer segments stained for blue cone opsin and avoided cones that did not. We conclude that the biocytin wide-field bipolar cell is an ON blue cone bipolar cell in the rabbit retina and is homologous to the blue cone bipolar cells that have been previously described in primate, mouse, and ground squirrel retinas [3].

In Vivo Rat Experiments [1]
\nTo test whether the newly designed tracers, L1 and L2, were more stable in vivo than conventional Biocytin (L), we performed iontophoretic injections of all three compounds into the cortex of 10 albino rats (Sprague−Dawley). Five rats (two with L, one with L1, and two with L2) were sacrificed after a survival time of 24 h, three rats (with L, L1, and L2) after a survival time of 96 h, and two rats (with L and L1) after 1 h.\n\nFor the experiments, 120 specimens of the fire-bellied toad Bombina orientalis were used. The animals were taken from a breeding colony at our institute. For the reconstruction of the anatomy of the telencephalon, 15-μm-thick transverse, horizontal and sagittal sections were made, embedded in paraffin, and counterstained with Klüver-Barrera. For the Biocytin labeling experiments, animals were deeply anesthetized in 0.5% tricaine methanesulfonate, cooled to a body temperature of 5°C, and perfused transcardially with 40 ml of ice-cold oxygenated Ringer's solution consisting of Na+ 100 mM, K+ 2 mM, Ca2+ 2 mM, Mg2+ 0.5 mM, Cl− 82 mM, HCO3− 25 mM, glucose 11 mM, buffered to a final pH of 7.3 through continuous perfusion of 95% O2 + 5% CO2. Brains were removed from the skull by a ventral approach.\n

\nAnterograde and retrograde labeling was made by application of Biocytin crystals to the brain. The isolated brains, either intact or longitudinally split into two halves, were exposed to the air and dried by using paper tissue, before small lesions were made with a glass micropipette at the site of application. The application sites and the number and mode of application are listed in Table 1. Ten minutes were allowed for the uptake of biocytin. Afterward, the brains were stored in Ringer's solution at room temperature for 4 hours and at 4°C for 16 to 32 hours. Brains were fixed in 2% paraformaldehyde and 2% glutaraldehyde, then 50-μm-thick transverse sections were cut on a Vibratome. Biocytin was visualized by means of an avidin-biotin-horseradish peroxidase complex using diaminobenzidine as chromogen with heavy-metal intensification (Adams, 1981). Sections were lightly counterstained with cresyl violet, dehydrated in ethanol, cleared in xylene, and cover-slipped.\n

\nFor intracellular labeling experiments, brains were split longitudinally, and half of the brain was fixed with stainless steel insect pins on the floor of a recording chamber (modified after Schaffer, 1982) with the cut medial surface pointing upward. The brain was continuously perfused with oxygenated Ringer's solution (6 ml/min) at a temperature of 14–18°C. The medial approach turned out to be favorable, because myelinated fibers below the lateral and ventral surfaces form a strong barrier to microelectrode penetrations. However, due to that method, possible contralateral projections of neurons could only be identified by their axons or axon collaterals entering the commissures. For intracellular labeling, micropipettes were filled with a 2% solution of Biocytin dissolved in 0.3 M potassium chloride. The impedance of the electrodes was 80–160 MΩ. The electrodes were advanced in steps of 1 or 2 μm, while a 200-msec hyperpolarizing current of 0.2 nA was applied every second. When a nerve cell membrane was penetrated, the potential dropped from −20 to −65 mV. For injection of Biocytin, a pulsed current of 1 nA was applied for 4 minutes. Usually, only one injection was done in each half of the brain. After the injection, brains were stored in oxygenated Ringer's solution at room temperature for 3 hours and at 4°C overnight. The brains were processed as described above for the anterograde and retrograde labeling experiments. Reconstructions of labeled neurons were made by hand with the aid of a camera lucida. Sections were scanned with a digital camera at a resolution of 3,900 × 3,090 pixels.[2]

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\nWide-field bipolar cells were selectively labeled in rabbit retinas by vitreal injections of Biocytin. Adult New Zealand White rabbits were first anesthetized with an intramuscular injection of ketamine (30 – 60 mg/kg) and xylazine (10 mg/kg) and then 20 μl of 5% Biocytin in ddH2O and 0.5% DMSO were injected into each eye. After 40 – 50 hour incubations, the rabbits were reanesthetized using the same agents and the eyes were ennucleated, hemisected and the retinas removed from the eye cup while immersed in oxygenated Ames Medium. Rabbits were euthanized with Euthosol (100 mg/kg, iv). Free floating retinas were fixed for 60 – 90 minutes in 4% formaldehyde and rinsed thoroughly in three changes of sodium phosphate buffer (0.1M, pH 7.4) over 30 minutes. For retinal wholemounts, tissue was stained immediately with antibodies. For retinal sections, tissue was embedded in 4% agarose (low melting point) and cut into 60 μm sections using a vibratome.\n

\nThe tissue was processed to reveal Biocytin next. Retinas were incubated overnight at 4°C in 0.25M Tris buffer with 0.5% Triton-X 100, rinsed in the same buffer the next day and then incubated for two days in avidin DN.. After rinsing, the retinas were incubated overnight in fluorescein anti-avidin D followed by thorough rinsing in buffer.\n

\nThe final step was to label all cones with peanut agglutinin, which labels certain disaccharides on the surface of outer and inner segments of all cones and in punctuate regions at the cone pedicles. Endogenous avidin and biotin in the retina was blocked by preincubating the tissue in an avidin/biotin blocking solution for 30 minutes at room temperature, rinsed in tris buffer and then incubated with biotinolated peanut agglutinin in the microwave at 150 watts for 13 minutes. The tissue was rinsed in sodium phosphate buffer (3 × 60 sec) and the lectin revealed with Texas Red conjugated strepavidin for 10 minutes, rinsed thoroughly in phosphate buffer and coverslipped in Vectashield shortly before viewing in the microscope.\n\nRetinal sections were processed using the same procedures except for the Biocytin step; incubation in avidin DN was shortened to overnight (rather than several days) and to two hours in fluorescein anti-avidin.\n

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\n\nImaging [3]
\nLabeled retinas were imaged with an Olympus Fluoview 300 Confocal microscope equipped with helium, argon and neon lasers and an Olympus 60 X water immersion objective (NA 1.2) at resolutions of 1024 × 1024 or 2048 × 2048. A series of optical sections in a single channel were collected every 0.5 μm in the inner plexiform layer to show the wide-field bipolar cell axons, in the outer plexiform layer to show the contacts made by the bipolar cell dendrites with overlying cones, and through the outer nuclear layer to link individual cone pedicles with corresponding outer segments. Each channel was scanned separately to reduce spectral cross-talk between channels. Comparisons of the cones, opsins and the Biocytin labeled cells were made by merging images of like focal planes using MetaVue (v 6.1) or Adobe PhotoShop and corrected for brightness and contrast using Adobe Photoshop.

[1]. Improved neuronal tract tracing with stable biocytin-derived neuroimaging agents. ACS Chem Neurosci. 2010 Feb 17;1(2):129-38.

[2]. Morphology and axonal projection pattern of neurons in the telencephalon of the fire-bellied toad Bombina orientalis: an anterograde, retrograde, and intracellular biocytin labeling study. J Comp Neurol. 2004 Oct 4;478(1):35-61.

[3]. Biocytin wide-field bipolar cells in rabbit retina selectively contact blue cones. J Comp Neurol. 2008 Jan 1;506(1):6-15.

Biocytin is a monocarboxylic acid amide formed by the condensation of the carboxylic acid group of biotin with the N(6)-amino group of L-lysine. It is a mouse metabolite. It is a derivative of azabicycloalkanes, thiabicycloalkanes, urea compounds, monocarboxylic acid amides, non-protein L-α-amino acids, and L-lysine. It is functionally related to biotin. It is a zwitterion tautomer of biocytin. Biocytin has been reported to exist in humans and rapeseed, and relevant data are available. In summary, we designed and synthesized two new stable tracers (L1 and L2) and evaluated their absorption and transport capabilities using common histochemical methods. The linkage between the biotin and lysine moieties in both molecules allows the new molecules to resist biotinylate cleavage. In vivo experiments on these synthesized biocytin derivatives demonstrated that they are indeed more stable conjugates. Even 96 hours after injection, staining of neuronal cell bodies and fibers at the injection site and distal terminal regions remained, while commercially available biocytin had almost degraded by then. Anterograde or retrograde transport was observed in the ipsilateral and contralateral cortex, striatum, thalamus, and further down the brainstem, indicating that they can be transported efficiently along axons. Therefore, L1 and L2 are ideal alternatives to traditional histological studies, avoiding the problems caused by endogenous degradation of tracer molecules before animal sacrifice. Importantly, the molecular design of these reagents can be easily diversified by conjugating different reporter molecules, serving as multimodal tracers. Thus, the reported molecules can serve as powerful tools for developing magnetic resonance imaging visualization reagents for in vivo studies of brain connectivity. [1]

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In this study, we used a combination of anterograde and retrograde labeling and intracellular biotin labeling because both methods have their advantages and disadvantages. Anterograde and retrograde labeling can quantitatively assess connectivity in specific brain regions well, but cannot provide detailed morphological information on individual neurons and their axonal projections. In addition, this method may lead to accidental damage to passing fibers, and neurons with very thick dendrites may be labeled with dendrites rather than axons. On the other hand, intracellular injection cannot provide quantitative information on projection strength and may be affected by differences in neuronal accessibility and penetration. For example, in Bombina orientalis, neurons in the medial, central, and lateral amygdala, as well as the ventral striatum and ventral globus pallidus, are relatively easily labeled via the medial approach through the longitudinal fissure; however, neurons in the lateral globus pallidus, SPTA, and dorsal striatum are more difficult to label because they can only be reached by passing through the ventricles. Labeling septal neurons is the most difficult because these cells are loosely arranged and can easily escape from the microelectrode tip. Despite these limitations, only a few significant differences were observed between anterograde, retrograde, and intracellular labeling results. Retrograde and partially anterograde labeling revealed projections from the ventrolateral cortex to the ventrolateral septum, ventral globus pallidus, nucleus accumbens, hypothalamus, parabrachial nucleus/visceral secondary nucleus, and medulla oblongata, but these projections were not revealed by intracellular labeling. Anterograde labeling also revealed projections from the SPTA to the optic tectum and hemianoid nucleus, and from the ventrolateral cortex to the posterior tubercle, projections not found in intracellular labeling. On the other hand, intracellular injection of nucleus accumbens cells (rather than anterograde tracing) revealed projections to the tegmentum and medulla oblongata. Some differences emerged when comparing anterograde and retrograde staining results, as biocytin appeared to outperform retrograde transport in our view. For example, fiber labeling was observed in the accessory olfactory bulb after applying the tracer to the medulla oblongata, but no retrogradely stained neurons were found in the medulla oblongata after the tracer was applied to the accessory olfactory bulb. This study did not delve deeply into the lateral cortex, which warrants further analysis. Our anterograde and retrograde tracing experiments revealed differences between the ventrolateral cortex and the dorsolateral cortex. It receives input from the main olfactory tract and, unlike the dorsolateral cortex, exhibits strong retrograde projection towards the main olfactory bulb. In the ventrolateral portion of the most caudal part of the ventrolateral cortex, near the caudal "ring" of the medial olfactory tract, a unique cell population connected to the adjacent caudal SPTA was labeled after biocytin labeling of the hypothalamus (see Figures 6 and 7). The anterior adjacent columnar structure, composed of the ventrolateral cortex and SPTA, shows only scattered retrograde labeled neurons. Based on their possible origin from inputs to the main olfactory bulb and projections to the hypothalamus, we believe that at least the caudally labeled neuronal clusters are homologous to the mammalian cortex or “olfactory” amygdala, as proposed by Scalia et al. (1991). [2]

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Biotin-stained wide-field bipolar cells are the cell type in the rabbit retina corresponding to the mammalian ON-type blue cone bipolar cells, which have been described in the retinas of monkeys, mice, and ground squirrels (Kouyama and Marshak, 1992; Haverkamp et al., 2005; Li and DeVries, 2006). Like their mammalian counterparts, rabbit wide-field cells are more than twice the size of any other type of bipolar cell (MacNeil et al., 2004). Their axons are located in the inner reticular layer 5, and are therefore considered ON-type bipolar cells. Furthermore, the dendrites of wide-field cells extend throughout the inner reticular layer and selectively contact cone cells expressing blue cone opsin in the outer segments, while avoiding cone cells that do not express blue cone opsin. We hypothesize that these contact points represent synaptic connections because we were able to trace the dendrites into the foot of the blue cone cell through a series of focused images and observe that they terminate near the PNA-marked region. Invaginated bipolar processes are a hallmark of ON-type bipolar cells, marking the site of synaptic formation between bipolar and cone cells (Hopkins and Boycott, 1995). Confocal studies of the cone cell foot showed that the PNA marker overlapped with the bason tube immunoreactive band and aligned with the Goα marker of ON-type bipolar cells (Haverkamp et al., 2001). This suggests that the proximity of the PNA marker to the biotinylated processes in the blue cone cell foot reflects synaptic relationships. Li and DeVries (2006) have clearly demonstrated synaptic communication between blue cone bipolar cells and blue cone cells in the ground squirrel retina. They confirmed that only one type of bipolar cell forms exclusive contact with blue cone cells, and that these contacts are functional; depolarization of presynaptic cone cells generates significant outward currents in coupled bipolar cells. [2]
In addition to the contacts formed between wide-field cell dendrites and cone cells, these dendrites also include small projections (Famiglietti, 1981) that fuse with the small dendrites of adjacent cells (Jeon and Masland, 1995). These projections typically originate from terminal clusters aligned with blue cone cells, have smooth surfaces, and do not connect to any cone cell feet that might suggest synaptic connections. Gap junctions may exist between the small dendrites of adjacent cells, but these cannot be confirmed in confocal images. [2]
A significant difference observed in the cone cell contacts of rabbit wide-field bipolar cells is the degree of divergence of photoreceptor input. In primates and mice, the density ratio of blue cone bipolar cells to blue cone cells is approximately 1:2, with each blue cone cell contacting multiple blue cone bipolar cells (Kouyama and Marshak, 1992; Kouyama and Marshak, 1997; Schein et al., 2004; Haverkamp et al., 2005). In rabbits, the distribution of blue cone cell inputs to each wide-area bipolar cell depends on the eccentricity of the retina. In the dorsal retina, the density of blue cone cells is lower relative to the number of wide-area bipolar cells, and blue cone cells contact the dendrites of multiple wide-area cells. In the ventral retina, multiple blue cone cells tend to converge onto each wide-area bipolar cell, and each blue cone cell contacts at most one cell (Fig. 7). The advantage of this arrangement is that it preserves the spatial representation of the blue signal in the ventral retina while better detecting the blue cone signal in the dorsal retina, where the number of blue cones is minimal. [2]

Dual-pigment cones[2]
In the rabbit peripheral ventral retina, a small subset of cones were simultaneously labeled with antibodies against both blue and red-green cone opsins. Mixed-opsin cones are common in the retinas of many mammals (Szél et al., 2000; Lukáts et al., 2005), but their specific function remains unclear. In the mouse retina, most cones express dual opsins (Applebury et al., 2000), but blue cone bipolar cells still form proper connections with true blue cones (Haverkamp et al., 2005), suggesting the existence of a color-dissociated blue channel in the mouse retina. In the rabbit retina, Biocytin wide-field cells contact all blue cone cells within their dendritic field, except at the ventral edge, where mixed-pigment cone cells are observed. At this eccentricity, the cell dendrites contact only a portion of the blue cone cells, suggesting that cone cells expressing multiple cone proteins are functionally distinct from true blue cone cells. The advantages of this hybrid cone cell structure are unclear, but it may be an adaptation that diurnal mammals evolved to better detect predators against a blue sky background (Ahnelt and Kolb, 2000; Peichl, 2005). [2]

Function of wide-field bipolar cells [2]
Biocytin The selectivity of wide-field bipolar cells to blue cone cells and their structural similarity to blue cone bipolar cells in the retinas of monkeys, mice, and ground squirrels suggest that they are involved in color processing in the rabbit retina. The idea that wide-field cells are selective for blue cone cells was first proposed by Familietti (1981). By counting cells and measuring the distance between the terminal clusters of wide-field bipolar dendrites, Familietti predicted that if wide-field cells were selective for blue cone cells, then the density of blue cone cells should increase toward the periphery. This prediction was confirmed by studies of cone cell labeling, which showed that the density of blue cone cells generally increases towards the ventral periphery (Juliusson et al., 1994; Famiglietti and Sharpe, 1995), and that wide-field cells in the ventral retina tend to contact more blue cone cells, which are then further away from the visual stripes. The ability of wide-field cells to selectively contact blue cone cells allows them to transmit spectrally separated signals to ganglion cells located in the inner retinal layer 5. [2]

There are still some questions about the identity of OFF-type blue cone bipolar cells in the rabbit retina. Famiglietti (1981) described two populations of wide-field bipolar cells in Golgi-stained tissue: one located in the b sublayer (wb type), which is likely biotin-labeled wide-field bipolar cells; and the other located in the a sublayer (wa type). wa cells have “extensive” axons of unknown length and narrow dendritic branches, consisting of 1 to 4 primary dendrites. Each dendrite contacts a cone cell, some of which are shared with wb cells. To date, no similar types have been found in other studies of rabbit bipolar cells (Mills and Massey, 1992; McGillem and Dacheux, 2001; MacNeil et al., 2004). The largest bipolar cells found in sublayer a of the retina are of the DAPI-Ba3 type (Mills and Massey, 1992), but their dendrites are more extensive than those of WA cells, with an average of 16 cone cells in contact per cell. Chiao and Liu (2006) injected a pair of OFF-type bipolar cells into the dorsal retina, which appeared to contact only blue cone cells. These cells had a narrow dendritic field with four main dendrites (similar to WA cells), each dendritic terminal aligned with the opsin staining direction of blue cone cells. However, the axonal field diameter of these cells was relatively small compared to the axons of wide-field biocytin bipolar cells (73 μm vs. >200 μm), so Chiao and Liu's cells are unlikely to be equivalent to WA cells. Further research is needed on OFF-type bipolar cells to elucidate the morphology of the OFF-type blue cone bipolar cell population. [2]

Biotin-labeled wide-field bipolar cell circuits [2]
Recordation of rabbit retinal ganglion cells showed the presence of color-antagonistic ganglion cells with blue light ON/green light OFF (Caldwell and Daw, 1978; De Monasterio, 1978; Vaney et al., 1981). The circuit mechanism of these light responses is unclear, but it is likely formed by the selective attachment of ON-type and OFF-type cone bipolar cells to a bilayer of ganglion cells, as observed in primates (Dacey and Lee, 1994; Calkins et al., 1998). G3 ganglion cells in the rabbit retina are ideal candidates for this attachment (Rockhill et al., 2002). It has a bilayer dendritic structure with the inner dendrites located at the junction of layers 4 and 5, close to the biotin-labeled wide-field cell axons, and the outer dendrites located in layer 2. We have confirmed that the dendrites of wide-field bipolar cells selectively contact blue cone cells in the outer reticular layer, whose axons are layered in the inner reticular layer 5. Combined with input from red-green cone cells of OFF-type bipolar cells located in the inner reticular layer A, G3 ganglion cells may be homologous to the blue-yellow cells described in primates (Calkins et al., 1998; Dacey, 2000). Another possibility is that wide-field bipolar cells can form synapses with monolayer ganglion cells located in the inner retinal layer 5, such as Ib2 (Famiglietti, 2004) or G10 ganglion cells (Rockhill et al., 2002). In a recent recording of blue cone cells in the primate retina, Packer et al. found that the blue-yellow spectral antagonism originates from the blue cone cells themselves (Packer et al., 2007). Therefore, bilayer ganglion cells are not a necessary condition for the color antagonistic responses recorded in rabbit retinal ganglion cells, which may be transmitted to the brain by monolayer ganglion cells. [3]

C16H28N4O4S

372.48

372.183

C, 51.59; H, 7.58; N, 15.04; O, 17.18; S, 8.61

576-19-2

576-19-2;

83814

White to off-white solid powder

1.2±0.1 g/cm3

748.0±60.0 °C at 760 mmHg

-245ºC (dec.)

406.2±32.9 °C

0.0±5.4 mmHg at 25°C

1.548

-1.02

5

6

11

25

491

4

S1C([H])([H])[C@@]2([H])[C@@]([H])([C@]1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[C@@]([H])(C(=O)O[H])N([H])[H])=O)N([H])C(N2[H])=O

BAQMYDQNMFBZNA-MNXVOIDGSA-N

InChI=1S/C16H28N4O4S/c17-10(15(22)23)5-3-4-8-18-13(21)7-2-1-6-12-14-11(9-25-12)19-16(24)20-14/h10-12,14H,1-9,17H2,(H,18,21)(H,22,23)(H2,19,20,24)/t10-,11-,12-,14-/m0/s1

N6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)-L-lysine

Biotinyl-L-lysine; Biocytin; 576-19-2; N'-Biotinyl-L-lysine; H-Lys(biotinyl)-OH; Biotinyl-L-lysine; epsilon-N-Biotinyl-L-lysine; Ne-Biotynyl-L-lysine; N-biotinyl-L-lysine; Biocytin

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)

H2O : ~50 mg/mL (~134.24 mM)

Solubility in Formulation 1: 100 mg/mL (268.47 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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