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Daspei

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DASPEI is a fluorescent dye used in biological research. It is a membrane-permeable stain that selectively labels mitochondria in living cells. DASPEI can be used to visualize and study the distribution and dynamics of mitochondria within cells.

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Lab products found in correlation

Calcein, by Merck Group (3 mentions) Dfc310fx color, by Leica (2 mentions) Application suite las software package, by Leica (2 mentions) Ms 222, by Merck Group (2 mentions) M205 fa, by Leica (1 mentions) M165 fc, by Leica (1 mentions) Low melting agarose, by Thermo Fisher Scientific (1 mentions) Eclipse te2000 u, by Nikon (1 mentions) D3418, by Merck Group (1 mentions) Smz 18 zoom stereo microscope, by Nikon (1 mentions) Tricaine 3 amino benzoic acidethylester, by Merck Group (1 mentions)

12 protocols using daspei

1

Live DASPEI Labeling in Fish

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2-[4-(Dimethylamino)styryl]-1-ethylpyridinium iodide (DASPEI) labeling in live fish was performed by diluting 1 mM DASPEI (Sigma-Aldrich) in system water. Fish were incubated in the solution for 1 min, quickly rinsed in system water and anesthetized for imaging. Images were taken with a Leica AF M205 FA stereomicroscope with the GFP2 filter. DASPEI cells were counted manually in ImageJ using the cell counter tool, because the tissue was too autofluorescent for quantification through the analyze particle option.
König D., Dagenais P., Senk A., Djonov V., Aegerter C.M, & Jaźwińska A. (2019). Distribution and Restoration of Serotonin-Immunoreactive Paraneuronal Cells During Caudal Fin Regeneration in Zebrafish. Frontiers in Molecular Neuroscience, 12, 227.
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2

Lateral Line Hair Cell Regeneration

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In order to study zfZNF32 function in lateral line system regeneration, hair cells in zebrafish larvae were damaged via neomycin treatment. ZfZNF−/− at 4 dpf were treated with 400 μM neomycin (Amersco, China) for 1 hr, rinsed 4 times in fresh water, and then returned to the incubator in normal embryo medium at 28.5°C. Wild-type zebrafish were used as a control [41 (link)].
A SOX2 morpholino (MO), 5′-GAA AGT CTA CCC CAC CAG CCG TAA A-32, was designed to block SOX2 gene expression. A standard MO (5′-CCT CTT ACC TCA GTT ACA ATT TAT A-32) was used as a control [42 (link)]. All MOs were purchased from Gene Tools. MOs were microinjected to zfZNF−/− and WT embryos at the one-cell stage, then the hair cells of 4 dpf larvae were damaged as the above.
2-[4-(dimethylamino) styryl]-N-ethylpyridiniym iodide (DASPEI, Sigma) was used to stain hair cells in lateral line neuromasts. After 12 and 24 hrs, larvae were placed into 0.005% DASPEI in embryo medium for 15 min and then anesthetized in MS222 (10 μg/ml, 3-aminobenzoic acid ethyl ester, methanesulfonate salt; Sigma) for 5 min. DASPEI-labeled cells in neuromasts were analyzed using fluorescence microscopy (ECLIPSE TE2000-U, Nikon, Japan) [43 (link)]. At the same time, larvae were collected for Q-PCR.
Wei Y., Li K., Yao S., Gao J., Li J., Shang Y., Zhang J., Zhang L., Li Y., Mo X., Meng W., Xiang R., Hu J., Lin P, & Wei Y. (2016). Loss of ZNF32 augments the regeneration of nervous lateral line system through negative regulation of SOX2 transcription. Oncotarget, 7(43), 70420-70436.
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3

Visualizing Neuromast Integrity in Fish

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To validate neuromast ablation following gentamicin treatment, fish were treated for 1 h with 0.05% DASPEI (2–4-dimethylaminostyryl-N-ethylpyridinium iodide) solution (Sigma Aldrich), a dye that specifically labels both superficial and canal neuromasts (Van Trump et al., 2010 (link)). Although the exact mechanism of DASPEI labeling is unknown, the dye is thought to enter cells through transduction channels and apical endocytosis, allowing its uptake by active hair cells via transduction-dependent mechanisms and making it highly specific for labeling intact neuromasts of the lateral line (Van Trump et al., 2010 (link)). After staining, fish were anesthetized and neuromasts were observed using a microscope (Leica M205 FA) set to 40 × magnification, 5.17 mm FOV, with a GFP filter set (excitation 450–490 nm). Photographs were captured with a high-resolution CCD camera (ProgRes C14) with ImagePro software (v.9.1). All images were acquired within ∼4 h of the end of baseline behavior recordings. All experimental fish were placed back in their home tanks and given approximately 24 h to recover before any further testing was done. Images represent tiled images merged in Photoshop CS6 (Adobe).
Lloyd E., Olive C., Stahl B.A., Jaggard J.B., Amaral P., Duboué E.R, & Keene A.C. (2018). Evolutionary shift towards lateral line dependent prey capture behavior in the blind Mexican cavefish. Developmental biology, 441(2), 328-337.
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4

Visualizing Neuromasts and Bone in Fish

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Thirty adult individuals were selected from our stock of surface fish, as well as 30 adult individuals from three different cave localities (Pachón, Tinaja, and Chica). Each individual was stained with Calcein (Sigma Aldrich; St. Louis, MO, USA) and 2-[4-(Dimethylamino)styryl]-1-ethylpyridinium iodide (DASPEI; Sigma Aldrich) in order to visualize bone and neuromasts, respectively. The left and right side of each fish was imaged simultaneously to visualize fluorescent bone and neuromast co-labels. A montage was created for each image by stacking a series of Z-plane images at multiple focal points. This resulted in all neuromast organs and bone margins to remain focused, despite slight variation in the Z-plane. All micrographs were collected using a Leica M205FA stereomicroscope (Wetzlar, Germany) equipped with a DFC310FX color camera. Montage images were collected utilizing the MultiFocus module within the Leica Application Suite (LAS) software package (version 3.8, Leica Microsystems, Buffalo Grove, IL, USA, 2015).
Gross J.B., Gangidine A, & Powers A.K. (2016). Asymmetric Facial Bone Fragmentation Mirrors Asymmetric Distribution of Cranial Neuromasts in Blind Mexican Cavefish. Symmetry, 8(11), 118.
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5

Assessing Cisplatin-Induced Hair Cell Toxicity

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Wild‐type (AB strain) zebrafish were grown to 5 days post‐fertilization (dpf) in standard E3 embryo media (Westerfield, 2000) and were bath‐treated with either 0, 5, 10, 25, or 50 μM of cisplatin in 6‐well plates, with 10–15 zebrafish larvae per well. After a 20‐h incubation with cisplatin at 28°C, wells were washed with embryo media before the fish were incubated in media containing 0.01% 2‐[4‐(dimethylamino) styryl]‐1‐ethylpyridinium iodide (DASPEI, Sigma‐Aldrich) to stain for neuromast mitochondrial activity for 20 min. Wells were washed again in embryo media and zebrafish larvae anesthetized with 4% tricaine. Neuromasts were imaged under a Leica M165 FC dissecting microscope equipped with a fluorescent filter. A standard scoring method for zebrafish hair cell viability was used (Chowdhury et al,2018): Five posterior lateral line (PLL) neuromasts for each fish were assigned a score representing cell viability based on DASPEI fluorescent intensity (2 for no noticeable decline, 1.5 for minor decline, 1 for moderate decline, 0.5 for severe decline, and 0 for complete loss of fluorescent intensity). These five scores were summed for each individual (10 = all hair cells appear normal and viable; 0 = intense ototoxicity).
Babolmorad G., Latif A., Domingo I.K., Pollock N.M., Delyea C., Rieger A.M., Allison W.T, & Bhavsar A.P. (2021). Toll‐like receptor 4 is activated by platinum and contributes to cisplatin‐induced ototoxicity. EMBO Reports, 22(5), e51280.
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6

DASPEI Staining of Zebrafish Larvae

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After microinjection, larvae (6 and 7 dpf) were immersed in 1 mM 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI; Sigma-Aldrich, Oakville, Ontario, Canada) in fish water for 1 hour. larvae were then oriented on their lateral side (anterior, left; posterior, right; dorsal, top) and mounted with methylcellulose in a depression slide for observation by fluorescence microscopy.
Gao X., Yuan Y.Y., Lin Q.F., Xu J.C., Wang W.Q., Qiao Y.H., Kang D.Y., Bai D., Xin F., Huang S.S., Qiu S.W., Guan L.P., Su Y., Wang G.J., Han M.Y., Jiang Y., Liu H.K, & Dai P. (2018). Mutation of IFNLR1, an interferon lambda receptor 1, is associated with autosomal-dominant non-syndromic hearing loss. Journal of Medical Genetics, 55(5), 298-306.
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7

Labeling Lateral Line Hair Cells

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Hair cells in neuromasts of the lateral line system were labeled by incubating live larvae in 5 μM 2-[4(Dimethylamino)styryl]-1-ethylpyridinium iodide (DASPEI, Sigma-Aldrich, St. Louis, MO) in Eggwater for 20 min. Larvae were washed 3 times in Eggwater and mounted in 1.2% low melting agarose (ThermoFisher, Waltham, MA) on the lid of a small Petri dish for visual inspection of the lateral line system and further dye injections.
Haehnel-Taguchi M., Fernandes A.M., Böhler M., Schmitt I., Tittel L, & Driever W. (2018). Projections of the Diencephalospinal Dopaminergic System to Peripheral Sense Organs in Larval Zebrafish (Danio rerio). Frontiers in Neuroanatomy, 12, 20.
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8

Visualizing Lateral Line Hair Cells

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After microinjection, larvae (5 dpf) were immersed in 1 mM DASPEI (2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide, Sigma) in fish water for 1 h, anesthetized with 0.016% (w/v) tricaine methanesulfonate (TMS) (MS-222, Sigma), oriented laterally (anterior, left; posterior, right; dorsal, top), and mounted (using methylcellulose) in a depression slide prior to fluorescence microscopy. Hair cells stereotypically located on the lateral line were stained as green dots.
Zhao W., Gao X., Qiu S., Gao B., Gao S., Zhang X., Kang D., Han W., Dai P, & Yuan Y. (2019). A subunit of V-ATPases, ATP6V1B2, underlies the pathology of intellectual disability. EBioMedicine, 45, 408-421.
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9

Visualizing Neuromasts and Bone in Fish

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Thirty adult individuals were selected from our stock of surface fish, as well as 30 adult individuals from three different cave localities (Pachón, Tinaja, and Chica). Each individual was stained with Calcein (Sigma Aldrich; St. Louis, MO, USA) and 2-[4-(Dimethylamino)styryl]-1-ethylpyridinium iodide (DASPEI; Sigma Aldrich) in order to visualize bone and neuromasts, respectively. The left and right side of each fish was imaged simultaneously to visualize fluorescent bone and neuromast co-labels. A montage was created for each image by stacking a series of Z-plane images at multiple focal points. This resulted in all neuromast organs and bone margins to remain focused, despite slight variation in the Z-plane. All micrographs were collected using a Leica M205FA stereomicroscope (Wetzlar, Germany) equipped with a DFC310FX color camera. Montage images were collected utilizing the MultiFocus module within the Leica Application Suite (LAS) software package (version 3.8, Leica Microsystems, Buffalo Grove, IL, USA, 2015).
Gross J.B., Gangidine A, & Powers A.K. (2016). Asymmetric Facial Bone Fragmentation Mirrors Asymmetric Distribution of Cranial Neuromasts in Blind Mexican Cavefish. Symmetry, 8(11), 118.
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10

Visualization of Zebrafish Lateral Line

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The lateral line begins to develop from neurogenic placodes at 1.5 days post fertilization or ~2.7mm SL (Hinaux et al. 2011 (link)), well before ossification of cranial bones. Neuromasts embedded in the infraorbital canal arise by 72 hours post fertilization (hpf) in zebrafish (Raible & Kruse 2000 (link)). Components of the lateral line, both canal and superficial neuromasts, can be visualized in vivo using 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI; Sigma Aldrich D3418; Raible & Kruse 2000 (link); Fig. 1A). Because DASPEI can be visualized under fluorescent light ranging from 488–600nm (green and red filters), we performed co-labeling for dermal facial bones using Calcein (Sigma Aldrich C0875) under the same light filter (GFP) at 488nm (Fig. 1A–F).
Juvenile fish were stained overnight in 2μM Calcein, buffered with NaOH in treated system water, in individual 1L tanks. Calcein is a compound that binds to calcified bony matrix (Jun Du et al. 2001 (link)) and is a more inclusive method for bone staining compared to other chromatic stains (i.e. Alizarin red). Following overnight staining, fish were placed in clean 1L tanks with system water to rinse for ~1 hour. Fish were then placed in individual glass bowls and immersed in a solution of 0.5μM DASPEI in system water for approximately 5 minutes.
Powers A.K., Boggs T.E, & Gross J.B. (2018). Canal neuromast position prefigures developmental patterning of the suborbital bone series in Astyanax cave- and surface-dwelling fish. Developmental biology, 441(2), 252-261.
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