Product overview
| Name | Janelia Fluor® 646 conjugation kit |
| Biological description | Overview The Hello Bio Janelia Fluor 646® conjugation kit allows the conjugation of antibodies and proteins to Janelia Fluor® 646 in as little at 90 minutes (15 minutes active time) with a high degree of labeling. There are many benefits of directly labeled proteins and antibodies such as:
Requirements
Pack size guidance Please note:
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| Description | Kit for conjugation of antibodies and other proteins to Janelia Fluor® 646 |
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Images
Figure 1. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining in rat cerebellum
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain neuronal projections in rat cerebellum. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured as a tilescan using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured using a 40x objective (DAPI: 4ms, Y5: 80ms exposures) in a z-stack (0.3µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining in rat hippocampus
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain neuronal projections in rat midbrain. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 5ms, Y5: 221ms exposures) in a z-stack (0.98µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining in rat cortex
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain neuronal projections in rat cortex. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured using a 20x objective (DAPI: 5ms, Y5: 221ms exposures) in a z-stack (0.6µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining in rat brainstem
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain axons in rat brainstem. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured using a 40x objective (DAPI: 10ms, Y5: 47ms exposures) in a z-stack (0.3µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining in rat brainstem
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain axons in rat brainstem. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured using a 40x objective (DAPI: 10ms, Y5: 60ms exposures) in a z-stack (0.3µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining of rat corpus callosum
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain the axons making up the corpus callosum. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 5ms, Y5: 221ms exposures) in a z-stack (0.6µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 1. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining in rat cerebellum
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain neuronal projections in rat cerebellum. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured as a tilescan using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured using a 40x objective (DAPI: 4ms, Y5: 80ms exposures) in a z-stack (0.3µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining in rat hippocampus
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain neuronal projections in rat midbrain. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 5ms, Y5: 221ms exposures) in a z-stack (0.98µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining in rat cortex
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain neuronal projections in rat cortex. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured using a 20x objective (DAPI: 5ms, Y5: 221ms exposures) in a z-stack (0.6µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining in rat brainstem
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain axons in rat brainstem. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured using a 40x objective (DAPI: 10ms, Y5: 47ms exposures) in a z-stack (0.3µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining in rat brainstem
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain axons in rat brainstem. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured using a 40x objective (DAPI: 10ms, Y5: 60ms exposures) in a z-stack (0.3µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Janelia Fluor® 646 conjugated anti-neurofilament light antibody staining of rat corpus callosum
Janelia Fluor® 646 was conjugated with HB7266 (rabbit monoclonal anti-neurofilament L antibody) following the provided protocol and used to stain the axons making up the corpus callosum. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight at 4°C in Janelia Fluor® 646 conjugated HB7266 at a 1:500 dilution. Following washing, DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei before sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (aqueous). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 5ms, Y5: 221ms exposures) in a z-stack (0.6µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Optical Data
| Fluorescence spectra |  |
| Emission color | Red |
| Max excitation wavelength | 646 nm |
| Max emission wavelength | 664 nm |
| Closest laser lines | 640nm |
| Spectrally similar dyes | Alexa Fluor® 647, Atto 647, DyLight® 650, Cyanine 5 |
| Quantum Yield (φ) | 0.54 |
| Extinction Coefficient (ε) | 152000 |
| Correction Factor 280 | 0.19 |
Biological Data
| Application notes | Conjugation Protocol Please follow this link to the conjugation protocol. Conjugation takes around 90 minutes with only 15 minutes of active time. |
Solubility & Handling
| Storage instructions | +4°C, except HB8351 at -20°C. Protect dye from moisture and light. |
| Important | This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use |
Chemical Data
| Kit contents |
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References for Janelia Fluor® 646 conjugation kit
References are publications that support the biological activity of the product
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Nanoscale segregation of channel and barrier claudins enables paracellular ion flux.
Gonschior H et al (2022) Nature communications 13 : 4985 -
Single-molecule localization microscopy.
Lelek M et al (2021) Nature reviews. Methods primers 1 :
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