Streptavidin Janelia Fluor® 646 is a biotin binding protein conjugated with the fluorescent dye Janelia Fluor® 646 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® 646 enabling immunofluorescent detection in IHC, ICC, flow cytometry and Western blot. Janelia Fluor® 646 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:
· Conjugated with Janelia Fluor® 646 (Ex: 652nm, Em: 675nm)
· Supplied as a more stable lyophilate
· Bright and photostable signal for repeated imaging
· Suited for IHC(IF), ICC, Western blotting and Flow cytometry
Description
Janelia Fluor® 646 conjugated streptavidin for detection and signal amplification of biotin coupled proteins and antibodies.
Figure 1. Parvalbumin positive interneurons and GFAP positive astrocytes in the rat hippocampus.
Mouse polyclonal anti-parvalbumin HB6457 and mouse monoclonal anti-GFAP HB6406 staining in the rat hippocampus. Parvalbumin was detected using an anti-mouse biotin antibody (HB11345) followed by incubation with Streptavidin Janelia Fluor® 646 (HB17045).
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 (1:1000 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® 646 (HB17045) 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 using a 20x objective in a z-stack with exposures: 1.0 µg/mL: DAP: 57.803 ms, GFP: 3061.716 ms, Y5: 1165.153 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. Calretinin and Parvalbumin expression in rat hippocampus.
Rabbit polyclonal anti-Calretinin HB6494 and mouse monoclonal anti-Parvalbumin HB6457 staining of different interneuron populations in the rat hippocampus. Calretinin was detected using an anti-rabbit biotin antibody (HB11036) followed by incubation with Streptavidin Janelia Fluor® 646. (HB17045).
Method: Rat brains were dissected and fixed overnight in 4% PFA before incubation 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-calretinin HB6494 (1:4,000 dilution) and anti-parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11036) at a dilution of 1:250, and goat anti-mouse Dylight™ 594 (Thermofisher) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646
(HB17045) 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 SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured using a 10x objective, 405nm (6.0% power, PMT: 725.1V gain), 580nm (3.63% power, Hyd: 68% gain) and 633nm (10.16% power, Hyd: 94.7% gain) laser lines in a z-stack (3.67µm spacing). The stack was 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® 646 (HB17045) staining in rat hippocampus.
Streptavidin Janelia Fluor® 646 (HB17045) produces optimal staining of parvalbumin positive interneurons in the hippocampus at 1 µg/mL.
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 (1:1,000 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® 646 (HB17045) at 0.5 µg/mL, 1.0 µg/mL, 5.0 µg/mL or 10 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:
0.5 µg/mL: DAP: 139.756 ms, Y5: 914.617 ms
1.0 µg/mL: DAP: 57.803 ms, Y5: 1165.153 ms
5.0 µg/mL: DAP: 139.756 ms, Y5: 914.617 ms
10.0 µg/mL: DAP: 139.756 ms, Y5: 431.117 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 4. Calretinin positive interneurons and astrocytes in the rat hippocampus.
abbit polyclonal anti-calretinin HB6494 staining interneurons and chicken polyclonal anti-GFAP a href="/anti-gfap-antibody-chicken-validab.html">HB6406 staining of astrocytes in the rat hippocampus. Calretinin was detected using an anti-rabbit biotin antibody (HB11036) followed by incubation with Streptavidin Janelia Fluor® 646. (HB17045).
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-calretinin HB6494 (1:1,000 dilution) and anti-GFAP HB6406 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11036 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® 646 (HB17045) 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 SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured 10x objective, 405nm (6.0% power, PMT: 725.1V gain), 488nm (2.18% power, Hyd: 22.9% gain) and 633nm (10.16% power, Hyd: 94.7% gain) laser lines in a z-stack (3.67µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Concentration response of Streptavidin Janelia Fluor® 646 (HB17045) staining in rat hippocampus.
Streptavidin Janelia Fluor® 646 (HB17045) produces optimal staining of Calretinin positive interneurons in the hippocampus at 1 µg/mL.
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 HB6494 (1:1,000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11345 at a dilution of 1:250. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 0.5 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:
0.5 µg/mL: DAP: 46.156 ms, Y5: 11222.92 ms
1.0 µg/mL: DAP: 46.156 ms, Y5: 6131.079 ms
5.0 µg/mL: DAP: .756 ms, Y5: 11222.92 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 positive interneurons and GFAP positive astrocytes in the rat hippocampus.
Mouse polyclonal anti-parvalbumin HB6457 and mouse monoclonal anti-GFAP HB6406 staining in the rat hippocampus. Parvalbumin was detected using an anti-mouse biotin antibody (HB11345) followed by incubation with Streptavidin Janelia Fluor® 646 (HB17045).
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 (1:1000 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® 646 (HB17045) 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 using a 20x objective in a z-stack with exposures: 1.0 µg/mL: DAP: 57.803 ms, GFP: 3061.716 ms, Y5: 1165.153 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. Calretinin and Parvalbumin expression in rat hippocampus.
Rabbit polyclonal anti-Calretinin HB6494 and mouse monoclonal anti-Parvalbumin HB6457 staining of different interneuron populations in the rat hippocampus. Calretinin was detected using an anti-rabbit biotin antibody (HB11036) followed by incubation with Streptavidin Janelia Fluor® 646. (HB17045).
Method: Rat brains were dissected and fixed overnight in 4% PFA before incubation 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-calretinin HB6494 (1:4,000 dilution) and anti-parvalbumin HB6457 (1:1000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11036) at a dilution of 1:250, and goat anti-mouse Dylight™ 594 (Thermofisher) at a dilution of 1:300. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646
(HB17045) 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 SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured using a 10x objective, 405nm (6.0% power, PMT: 725.1V gain), 580nm (3.63% power, Hyd: 68% gain) and 633nm (10.16% power, Hyd: 94.7% gain) laser lines in a z-stack (3.67µm spacing). The stack was 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® 646 (HB17045) staining in rat hippocampus.
Streptavidin Janelia Fluor® 646 (HB17045) produces optimal staining of parvalbumin positive interneurons in the hippocampus at 1 µg/mL.
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 (1:1,000 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® 646 (HB17045) at 0.5 µg/mL, 1.0 µg/mL, 5.0 µg/mL or 10 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:
0.5 µg/mL: DAP: 139.756 ms, Y5: 914.617 ms
1.0 µg/mL: DAP: 57.803 ms, Y5: 1165.153 ms
5.0 µg/mL: DAP: 139.756 ms, Y5: 914.617 ms
10.0 µg/mL: DAP: 139.756 ms, Y5: 431.117 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 4. Calretinin positive interneurons and astrocytes in the rat hippocampus.
abbit polyclonal anti-calretinin HB6494 staining interneurons and chicken polyclonal anti-GFAP a href="/anti-gfap-antibody-chicken-validab.html">HB6406 staining of astrocytes in the rat hippocampus. Calretinin was detected using an anti-rabbit biotin antibody (HB11036) followed by incubation with Streptavidin Janelia Fluor® 646. (HB17045).
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-calretinin HB6494 (1:1,000 dilution) and anti-GFAP HB6406 (1:4000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11036 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® 646 (HB17045) 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 SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured 10x objective, 405nm (6.0% power, PMT: 725.1V gain), 488nm (2.18% power, Hyd: 22.9% gain) and 633nm (10.16% power, Hyd: 94.7% gain) laser lines in a z-stack (3.67µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Concentration response of Streptavidin Janelia Fluor® 646 (HB17045) staining in rat hippocampus.
Streptavidin Janelia Fluor® 646 (HB17045) produces optimal staining of Calretinin positive interneurons in the hippocampus at 1 µg/mL.
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 HB6494 (1:1,000 dilution) at 4°C. This was followed by a two hour incubation with goat anti-rabbit biotin antibody HB11345 at a dilution of 1:250. Following three washes in PBST, sections were incubated with Streptavidin Janelia Fluor® 646 (HB17045) at 0.5 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:
0.5 µg/mL: DAP: 46.156 ms, Y5: 11222.92 ms
1.0 µg/mL: DAP: 46.156 ms, Y5: 6131.079 ms
5.0 µg/mL: DAP: .756 ms, Y5: 11222.92 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).
#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® 646 in blocking buffer for 2 hours.
7. Wash sections three time 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 640nm laser or Cy5 filter set to excite Streptavidin Janelia Fluor® 646.
Solubility & Handling
Storage instructions
-20°C then use reconstitution advice
Reconstitution advice
We recommend reconstituting with either 1ml of:
dH2O and storing at 4°C
50:50 ratio of dH2O to glycerol and storing at -20°C
dH2O then aliquot and store at -80°C
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.
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