Biotin Tyramide Signal Amplification Kit - Janelia Fluor® 525
Product overview
| Name | Biotin Tyramide Signal Amplification Kit - Janelia Fluor® 525 |
| Biological description | Biotin Tyramide Signal Amplification Kit – Janelia Fluor® 525 enables immunofluorescent detection of low abundance targets with a bright and photostable fluorophore. Biotin Tyramide Signal Amplification Kits enhance signal through deposition of biotin tyramide on neighbouring molecules. Janelia Fluor® 525 detects biotin with extremely high affinity, resulting in increased sensitivity and signal detection. 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.
This kit provides the reagents for 100 x 1mL reactions, with a protocol that can easily be adapted for immunohistochemistry (IHC), immunocytochemistry (ICC) and in situ hybridisation (ISH).
Key features:
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| Description | Immunofluorescent signal amplification kit using biotin tyramide and Streptavidin Janelia Fluor® 525. |
Images
Figure 1: Tyramide Signal Amplification compared to detection using secondary biotin antibody and streptavidin
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 anti-parvalbumin HB6457 (1:4,000 dilution) at 4°C.
Sections were incubated with HB11345 at 1:250 for two hours, washed, and incubated with Streptavidin Janelia Fluor® 525 HB15382 to detect parvalbumin (right hand side image).
Tyramide Signal Amplification Kit – Janelia Fluor® 525 (HB19791) was used to detect parvalbumin on the lefthand side image. Sections were incubated with goat anti-mouse HRP antibody (Sigma) at 1:2000 dilution for two hours. Following three washes in PBST, sections were incubated with 1:1000 dilution of biotin tyramide, 0.003% hydrogen peroxide and 0.1% Tween20 in PBS for 15 minutes. The amplification reaction was quenched using stop solution for 10 minutes. Following three washes in PBST, sections were incubated with 1:1000 dilution of Streptavidin Janelia Fluor® 525 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 and TSA protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: DAP:11.851 ms, Y3: 650.367 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 expression in the rat cerebellum using tyramide signal amplification.
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 anti-parvalbumin HB6457 (1:4,000 dilution) at 4°C. Sections were incubated with goat anti-mouse HRP antibody (Sigma) at 1:2000 dilution for two hours.
Tyramide Signal Amplification Kit – Janelia Fluor® 525 (HB19791) was used to detect parvalbumin: following three washes in PBST, sections were incubated with 1:1000 dilution of biotin tyramide, 0.003% hydrogen peroxide and 0.1% Tween20 in PBS for 15 minutes. The amplification reaction was quenched using stop solution for 10 minutes. Following three washes in PBST, sections were incubated with 1:1000 dilution of Streptavidin Janelia Fluor® 525 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 and TSA protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: DAP: 100 ms, GFP: 421.117 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: Parvalbumin positive neurons in the rat hippocampus, detected using tyramide signal amplification
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 anti-parvalbumin HB6457 (1:4,000 dilution) at 4°C. Sections were incubated with goat anti-mouse HRP antibody (Sigma) at 1:2000 dilution for two hours.
Tyramide Signal Amplification Kit – Janelia Fluor® 525 (HB19791) was used to detect parvalbumin: following three washes in PBST, sections were incubated with 1:1000 dilution of biotin tyramide, 0.003% hydrogen peroxide and 0.1% Tween20 in PBS for 15 minutes. The amplification reaction was quenched using stop solution for 10 minutes. Following three washes in PBST, sections were incubated with 1:1000 dilution of Streptavidin Janelia Fluor® 525 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 and TSA protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: DAP: 50 ms, Y3: 597.756 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 4: Tyramide signal amplification of parvalbumin positive interneurons in the 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 in anti-parvalbumin HB6457 (1:4,000 dilution) at 4°C. Sections were incubated with goat anti-mouse HRP antibody (Sigma) at 1:2000 dilution for two hours.
Tyramide Signal Amplification Kit – Janelia Fluor® 525 (HB19791) was used to detect parvalbumin: following three washes in PBST, sections were incubated with 1:1000 dilution of biotin tyramide, 0.003% hydrogen peroxide and 0.1% Tween20 in PBS for 15 minutes. The amplification reaction was quenched using stop solution for 10 minutes. Following three washes in PBST, sections were incubated with 1:1000 dilution of Streptavidin Janelia Fluor® 525 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 and TSA protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: DAP: 100 ms, GFP: 421.117 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: Tyramide Signal Amplification compared to detection using secondary biotin antibody and streptavidin
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 anti-parvalbumin HB6457 (1:4,000 dilution) at 4°C.
Sections were incubated with HB11345 at 1:250 for two hours, washed, and incubated with Streptavidin Janelia Fluor® 525 HB15382 to detect parvalbumin (right hand side image).
Tyramide Signal Amplification Kit – Janelia Fluor® 525 (HB19791) was used to detect parvalbumin on the lefthand side image. Sections were incubated with goat anti-mouse HRP antibody (Sigma) at 1:2000 dilution for two hours. Following three washes in PBST, sections were incubated with 1:1000 dilution of biotin tyramide, 0.003% hydrogen peroxide and 0.1% Tween20 in PBS for 15 minutes. The amplification reaction was quenched using stop solution for 10 minutes. Following three washes in PBST, sections were incubated with 1:1000 dilution of Streptavidin Janelia Fluor® 525 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 and TSA protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: DAP:11.851 ms, Y3: 650.367 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 expression in the rat cerebellum using tyramide signal amplification.
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 anti-parvalbumin HB6457 (1:4,000 dilution) at 4°C. Sections were incubated with goat anti-mouse HRP antibody (Sigma) at 1:2000 dilution for two hours.
Tyramide Signal Amplification Kit – Janelia Fluor® 525 (HB19791) was used to detect parvalbumin: following three washes in PBST, sections were incubated with 1:1000 dilution of biotin tyramide, 0.003% hydrogen peroxide and 0.1% Tween20 in PBS for 15 minutes. The amplification reaction was quenched using stop solution for 10 minutes. Following three washes in PBST, sections were incubated with 1:1000 dilution of Streptavidin Janelia Fluor® 525 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 and TSA protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: DAP: 100 ms, GFP: 421.117 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: Parvalbumin positive neurons in the rat hippocampus, detected using tyramide signal amplification
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 anti-parvalbumin HB6457 (1:4,000 dilution) at 4°C. Sections were incubated with goat anti-mouse HRP antibody (Sigma) at 1:2000 dilution for two hours.
Tyramide Signal Amplification Kit – Janelia Fluor® 525 (HB19791) was used to detect parvalbumin: following three washes in PBST, sections were incubated with 1:1000 dilution of biotin tyramide, 0.003% hydrogen peroxide and 0.1% Tween20 in PBS for 15 minutes. The amplification reaction was quenched using stop solution for 10 minutes. Following three washes in PBST, sections were incubated with 1:1000 dilution of Streptavidin Janelia Fluor® 525 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 and TSA protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: DAP: 50 ms, Y3: 597.756 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 4: Tyramide signal amplification of parvalbumin positive interneurons in the 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 in anti-parvalbumin HB6457 (1:4,000 dilution) at 4°C. Sections were incubated with goat anti-mouse HRP antibody (Sigma) at 1:2000 dilution for two hours.
Tyramide Signal Amplification Kit – Janelia Fluor® 525 (HB19791) was used to detect parvalbumin: following three washes in PBST, sections were incubated with 1:1000 dilution of biotin tyramide, 0.003% hydrogen peroxide and 0.1% Tween20 in PBS for 15 minutes. The amplification reaction was quenched using stop solution for 10 minutes. Following three washes in PBST, sections were incubated with 1:1000 dilution of Streptavidin Janelia Fluor® 525 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 and TSA protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. Images were captured using a 20x objective in a z-stack with exposures: DAP: 100 ms, GFP: 421.117 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 nm |
| Max emission wavelength | 549 nm |
| 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) | 122,000 M-1cm-1 |
Biological Data
| Application notes | Biotin Tyramide Signal Amplification Protocol Please follow this link to the amplification protocol. |
Solubility & Handling
| Storage instructions | -20°C |
| Reconstitution advice | To reconstitute Streptavidin JF525 (HB15382) we recommend reconstituting with either 100 µl of:
Take care when opening as the precipitate is extremely light and can easily be lost if disturbed. When reconstituting make sure that the streptavidin is thoroughly dissolved by pipetting up and down before giving the streptavidin a brief spin at <10,000g to make sure that all material is recovered and at the bottom of the tube. To reconstitute Biotin Tyramide (HB3262) add 100 µL of DMSO. Aliquot and store at -20°C. |
| Storage buffer | Streptavidin JF525 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
| Licensing details | Sold under license from the Howard Hughes Medical Institute, Janelia Research Campus |
| Kit contents | Supplied components:
The following components are required to perform this assay but are not included in the kit:
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Technical guides
Biotin Tyramide Signal Amplification Kit ProtocolReferences for Biotin Tyramide Signal Amplification Kit - Janelia Fluor® 525
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Tyramide Signal Amplification for Immunofluorescent Enhancement.
Faget L et al (2015) Methods in molecular biology (Clifton, N.J.) 1318 : 161-72 -
Application of tyramide signal amplification system to immunohistochemistry: a potent method to localize antigens that are not detectable by ordinary method.
Toda Y et al (1999) Pathology international 49 : 479-83 -
Amplification of fluorescent in situ hybridisation signals in formalin fixed paraffin wax embedded sections of colon tumour using biotinylated tyramide.
McKay JA et al (1997) Molecular pathology : MP 50 : 322-5
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