Streptavidin Janelia Fluor® 525
Product overview
| Name | Streptavidin Janelia Fluor® 525 |
| Biological description | Streptavidin Janelia Fluor® 525 is a biotin binding protein conjugated with the fluorescent dye Janelia Fluor® 525 and can be used to detect biotin labelled molecules such as nucleic acids, antibodies, and other proteins. Biotinylated antibodies are bound with extremely high affinity by Streptavidin Janelia Fluor® 525 enabling immunofluorescent detection in IHC, ICC, flow cytometry and Western blot. Janelia Fluor® 525 and the other members of the Janelia Fluor® family are bright and highly photostable fluorophores particularly suited for super resolution imaging such as dSTORM and STED.
Key features:
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| Description | Janelia Fluor® 525 conjugated streptavidin for detection and signal amplification of biotin coupled proteins and antibodies. |
Images
Figure 1. Parvalbumin and Calretinin expressing interneurons in the rat hippocampus.
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 in Parvalbumin HB6457 (1:1000 dilution) and Calretinin HB6494 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody at a dilution of 1:250 and goat anti-rabbit Dylight™ 650 (Thermofisher) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 525 (HB15382) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured as a tilescan using a 20x objective in a z-stack with exposures: A4: 57.803 ms, Y3: 599.044 ms, Y5 599.044 ms. Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Parvalbumin expressing interneurons and GFAP expressing astrocytes in the rat hippocampus.
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 in Parvalbumin HB6457 (1:1000 dilution) and GFAP HB6406 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody HB11345 at a dilution of 1:250 and goat anti-chicken Alexa Fluor™ 488 (Invitrogen) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 525 (HB15382) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1 µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: A4: 57.803 ms, GFP: 524.511 ms, Y3: 524.211 ms Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Concentration response of Streptavidin Janelia Fluor® 525 in detecting Parvalbumin expressing neurons in the rat hippocampus.
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 in Parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody HB11345 at a dilution of 1:250. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 525 (HB15382) at 0.25 µg/mL, 1.0 µg/mL or 5.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures:
- 0.25 µg/mL: A4: 139.756 ms, Y3: 1821.082 ms
- 1.0 µg/mL: A4: 57.803 ms, Y3: 599.044 ms
- 5.0 µg/mL: A4: 146.669 ms, Y3: 1276.088 ms
Figure 4: Streptavidin Janelia Fluor® 525 (HB15382) detection of parvalbumin positive interneurons in the rat hippocampus
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 in Parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody HB11345 at a dilution of 1:250. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 525 (HB15382) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1 µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: A4: 54.669 ms, Y3: 599.044 ms Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 1. Parvalbumin and Calretinin expressing interneurons in the rat hippocampus.
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 in Parvalbumin HB6457 (1:1000 dilution) and Calretinin HB6494 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody at a dilution of 1:250 and goat anti-rabbit Dylight™ 650 (Thermofisher) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 525 (HB15382) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured as a tilescan using a 20x objective in a z-stack with exposures: A4: 57.803 ms, Y3: 599.044 ms, Y5 599.044 ms. Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Parvalbumin expressing interneurons and GFAP expressing astrocytes in the rat hippocampus.
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 in Parvalbumin HB6457 (1:1000 dilution) and GFAP HB6406 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody HB11345 at a dilution of 1:250 and goat anti-chicken Alexa Fluor™ 488 (Invitrogen) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 525 (HB15382) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1 µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: A4: 57.803 ms, GFP: 524.511 ms, Y3: 524.211 ms Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Concentration response of Streptavidin Janelia Fluor® 525 in detecting Parvalbumin expressing neurons in the rat hippocampus.
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 in Parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody HB11345 at a dilution of 1:250. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 525 (HB15382) at 0.25 µg/mL, 1.0 µg/mL or 5.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures:
- 0.25 µg/mL: A4: 139.756 ms, Y3: 1821.082 ms
- 1.0 µg/mL: A4: 57.803 ms, Y3: 599.044 ms
- 5.0 µg/mL: A4: 146.669 ms, Y3: 1276.088 ms
Figure 4: Streptavidin Janelia Fluor® 525 (HB15382) detection of parvalbumin positive interneurons in the rat hippocampus
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 in Parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-mouse biotin antibody HB11345 at a dilution of 1:250. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 525 (HB15382) at 1.0 µg/mL for 2 hours at room temperature. DAPI HB0747 was used at 1 µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: A4: 54.669 ms, Y3: 599.044 ms Stacks were deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Optical Data
| Fluorescence spectra |  |
| Emission color | Yellow |
| Max excitation wavelength | 525 |
| Max emission wavelength | 549 |
| Closest laser lines | 532nm |
| Spectrally similar dyes | Alexa Fluor® 514, Alexa Fluor® 532, Atto 532, CF532, CF514 |
| Quantum Yield (φ) | 0.91 |
| Extinction Coefficient (ε) | 122000 M-1cm-1 |
Biological Data
| Application notes | #Protocol 1: Detecting biotin-labelled antibodies in IHC 1. Incubate free floating rat brain sections (40µm) in sodium borohydride (NaBH4) for 15 minutes followed by 2 hours in blocking buffer (0.05M glycine, 2% BSA and 3% donkey serum). 2. Incubate sections with primary antibody in blocking buffer at 4°C overnight, as in our IHC protocol. 3. Wash sections three times in PBST for 5 minutes each. 4. Incubate sections with 2 µg/mL goat anti-mouse biotin antibody HB11345 or goat anti-rabbit antibody HB11036 diluted in blocking buffer for 2 hours at RT. 5. Wash sections three times in PBST for 5 minutes each. 6. Incubate sections with 1 µg/mL Streptavidin Janelia Fluor® 525 in blocking buffer for 2 hours. 7. Wash sections three times in PBST for 5 minutes each. 8. Incubate sections with 10 µg/mL DAPI for 10 minutes. 9. Wash sections in dH2O, mount on glass slides with mounting media and cover with coverslip. 10. Image the sections on a microscope using a 532nm laser or TRITC filter set to excite Streptavidin Janelia Fluor® 525. |
Solubility & Handling
| Storage instructions | -20°C then use reconstitution advice |
| Reconstitution advice | We recommend reconstituting with either 1ml of:
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| Storage buffer | When reconstituted contains PBS with 0.05% sodium azide and 1% recombinant BSA. |
| 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
| Molecular Weight | 52 |
| Formulation | Lyophilised. When reconstituted contains 0.05% sodium azide and 1% recombinant albumin |
| Licensing details | Sold under license from the Howard Hughes Medical Institute, Janelia Research Campus |
References for Streptavidin Janelia Fluor® 525
-
Recent advances in the engineering and application of streptavidin-like molecules.
Le Q et al (2019) Applied microbiology and biotechnology 103 : 7355-7365 -
Streptavidin-biotin technology: improvements and innovations in chemical and biological applications.
Dundas CM et al (2013) Applied microbiology and biotechnology 97 : 9343-53 -
Essentials of biorecognition: the (strept)avidin-biotin system as a model for protein-protein and protein-ligand interaction.
Wilchek M et al (2006) Immunology letters 103 : 27-32
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