Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed ValidAb™
(HB8556)
Product overview
| Name | Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed ValidAb™ |
| Host | Goat |
| Clonality | Polyclonal |
| Target | Rabbit IgG H&L |
| Conjugate | Janelia Fluor® 549 |
| Description | Goat Anti-Rabbit IgG H&L Janelia Fluor® 549 secondary antibody. Part of the ValidAb™ range of highly validated, data-rich antibodies. |
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Validation data
Figure 1. Neurofilament L staining in rat brain using HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed secondary antibody.
Rat brain stained for Neurofilament L (NFL, HB7266) using HB8556 goat anti-rabbit IgG H&L (Janelia Fluor® 549) preadsorbed secondary antibody. 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% goat serum before incubation overnight in HB7266 (1:2,000, 0.5µg/ml). This was followed by a two hour incubation with HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMi8 confocal microscope in Lightning mode with a 40x objective using 405nm (30.1% power, PMT: 553.8V gain) and 496nm (0.5% power, Hyd: 10.0% gain) lasers. Following capture, the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Neurofilament L staining in rat hippocampus using HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed secondary antibody.
Rat hippocampus stained for Neurofilament L (NFL, HB7266) and NeuN (HB6429) using HB8556 goat anti-rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies. 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% goat serum before incubation overnight in HB7266 (1:2,000 dilution, 0.5µg/ml) and HB6429 (1:1,000 dilution, 1µg/ml). This was followed by a two hour incubation with HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMI6000B fluorescence microscope with a 20x objective using DAP (8ms exposure), L5 (50ms exposure) and RHO (100ms exposure) filters. Following capture, the image was deconvolved in Huygens Professional then the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Neurofilament L staining in rat cortex using HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed secondary antibody.
Rat cortex stained for Neurofilament L (NFL, HB7266) and NeuN (HB6429) using HB8556 goat anti-rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies. 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% goat serum before incubation overnight in HB7266 (1:2,000 dilution, 0.5µg/ml) and HB6429 (1:1,000 dilution, 1µg/ml). This was followed by a two hour incubation with HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMi8 confocal microscope in Lightning mode with a 20x objective using 405nm (30.1% power, PMT: 708V gain), 488nm (3.0% power, Hyd: 32.1% gain) and 561nm (1.5% power, 109% gain) lasers. Following capture 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® 549 conjugated secondary antibodies show superior antifade performance compared to those conjugated with DyLight 550
Janelia Fluor® 549 conjugated secondary antibodies show greater resistance to photobleaching than DyLight 550 conjugated secondaries in a HEK293T based cell assay. Method: HEK293T cells were cultured on 10mm coverslips and stained with HB6491 (1:8,000 dilution, 125ng/ml) and either a goat anti-mouse DyLight 550 or goat anti-mouse Janelia Fluor® 549 conjugated secondary antibody. For more information please see our ICC protocol. Imaging was conducted with a Leica SPE confocal laser scanning microscope where cells were repeatedly imaged with the 488nm laser at 100% power over 30 seconds of continuous exposure to induce photobleaching. Average intensity values were calculated from images then data was normalised to the first exposure frame to generate normalised intensity. Statistics: decay curve: F-test, F5,350=1685, p<0.0001, final intensity: two tailed t-test, t16=19.15, p<0.0001.
Figure 5. Neurofilament L staining in rat cortex using HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed secondary antibody.
Rat cortex stained for Neurofilament L (NFL, HB7266) and NeuN (HB6429) using HB8556 goat anti-rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies. 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% goat serum before incubation overnight in HB7266 (1:2,000 dilution, 0.5µg/ml) and HB6429 (1:1,000 dilution, 1µg/ml). This was followed by a two hour incubation with HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMI6000B fluorescence microscope with a 20x objective using DAP (8ms exposure), L5 (50ms exposure) and RHO (100ms exposure) filters. Following capture, the image was deconvolved in Huygens Professional then the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 1. Neurofilament L staining in rat brain using HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed secondary antibody.
Rat brain stained for Neurofilament L (NFL, HB7266) using HB8556 goat anti-rabbit IgG H&L (Janelia Fluor® 549) preadsorbed secondary antibody. 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% goat serum before incubation overnight in HB7266 (1:2,000, 0.5µg/ml). This was followed by a two hour incubation with HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMi8 confocal microscope in Lightning mode with a 40x objective using 405nm (30.1% power, PMT: 553.8V gain) and 496nm (0.5% power, Hyd: 10.0% gain) lasers. Following capture, the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Neurofilament L staining in rat hippocampus using HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed secondary antibody.
Rat hippocampus stained for Neurofilament L (NFL, HB7266) and NeuN (HB6429) using HB8556 goat anti-rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies. 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% goat serum before incubation overnight in HB7266 (1:2,000 dilution, 0.5µg/ml) and HB6429 (1:1,000 dilution, 1µg/ml). This was followed by a two hour incubation with HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMI6000B fluorescence microscope with a 20x objective using DAP (8ms exposure), L5 (50ms exposure) and RHO (100ms exposure) filters. Following capture, the image was deconvolved in Huygens Professional then the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Neurofilament L staining in rat cortex using HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed secondary antibody.
Rat cortex stained for Neurofilament L (NFL, HB7266) and NeuN (HB6429) using HB8556 goat anti-rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies. 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% goat serum before incubation overnight in HB7266 (1:2,000 dilution, 0.5µg/ml) and HB6429 (1:1,000 dilution, 1µg/ml). This was followed by a two hour incubation with HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMi8 confocal microscope in Lightning mode with a 20x objective using 405nm (30.1% power, PMT: 708V gain), 488nm (3.0% power, Hyd: 32.1% gain) and 561nm (1.5% power, 109% gain) lasers. Following capture 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® 549 conjugated secondary antibodies show superior antifade performance compared to those conjugated with DyLight 550
Janelia Fluor® 549 conjugated secondary antibodies show greater resistance to photobleaching than DyLight 550 conjugated secondaries in a HEK293T based cell assay. Method: HEK293T cells were cultured on 10mm coverslips and stained with HB6491 (1:8,000 dilution, 125ng/ml) and either a goat anti-mouse DyLight 550 or goat anti-mouse Janelia Fluor® 549 conjugated secondary antibody. For more information please see our ICC protocol. Imaging was conducted with a Leica SPE confocal laser scanning microscope where cells were repeatedly imaged with the 488nm laser at 100% power over 30 seconds of continuous exposure to induce photobleaching. Average intensity values were calculated from images then data was normalised to the first exposure frame to generate normalised intensity. Statistics: decay curve: F-test, F5,350=1685, p<0.0001, final intensity: two tailed t-test, t16=19.15, p<0.0001.
Figure 5. Neurofilament L staining in rat cortex using HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed secondary antibody.
Rat cortex stained for Neurofilament L (NFL, HB7266) and NeuN (HB6429) using HB8556 goat anti-rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies. 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% goat serum before incubation overnight in HB7266 (1:2,000 dilution, 0.5µg/ml) and HB6429 (1:1,000 dilution, 1µg/ml). This was followed by a two hour incubation with HB8556 Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed and goat anti-mouse DyLight 488 conjugated secondary antibodies at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMI6000B fluorescence microscope with a 20x objective using DAP (8ms exposure), L5 (50ms exposure) and RHO (100ms exposure) filters. Following capture, the image was deconvolved in Huygens Professional then the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Product information
| Immunogen | Purified rabbit IgG |
| Purification notes | Immunogen affinity chromatography. Pre-adsorbed with human, mouse and rat serum proteins |
| Concentration | 1mg/ml |
| Formulation | 20% glycerol in PBS with 0.05% sodium azide and 1% recombinant albumin |
Tested applications
| Applications | ELISA, FACS and flow cytometry, ICC, live cell imaging, IHC(IF) |
| IHC(IF) optimal concentration | 1:300 to 1:2,000 dilution (0.5 - 3.3µg/ml). Optimise dependent upon assay. A good starting point is 1:500 (2µg/ml). |
| ICC optimal concentration | 1:300 to 1:2,000 dilution (0.5 - 3.3µg/ml). Optimise dependent upon assay. A good starting point is 1:500 (2µg/ml). |
| Negative control | While this antibody has been cross-adsorbed to reduce non-specific binding it is still often worthwhile to conduct a control experiment where the primary antibody is omitted to give confidence that the staining pattern observed is specific. |
Optical Data
| Max excitation wavelength | 549nm |
| Max emission wavelength | 571nm |
| Emission color | Yellow |
| Closest laser lines | 561nm |
| Spectrally similar dyes | DyLight 550, Alexa Fluor® 546, Alexa Fluor® 555, BDY TMR-X, Atto 550, Cyanine 3, CF 555, TAMRA |
| Fluorescence spectra |  |
Storage & Handling
| Storage instructions | +4°C |
| Important | This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use |
Technical guides
Western Blot Protocol (1 MB) Immunocytochemistry Protocol (1.2 MB) Immunohistochemistry Protocol (1.6 MB) References for Goat Anti-Rabbit IgG H&L (Janelia Fluor® 549) preadsorbed ValidAb™
References are publications that support the biological activity of the product
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Single-molecule localization microscopy.
Lelek M et al (2021) Nature reviews. Methods primers 1 : -
Precision of tissue patterning is controlled by dynamical properties of gene regulatory networks.
Exelby K et al (2021) Development (Cambridge, England) 148 :
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