Goat Anti-Rabbit IgG H&L (AF488) preadsorbed ValidAb™
(HB9317)
Product overview
| Name | Goat Anti-Rabbit IgG H&L (AF488) preadsorbed ValidAb™ |
| Host | Goat |
| Clonality | Polyclonal |
| Target | Rabbit IgG H&L |
| Conjugate | AF488 |
| Description | Goat Anti-Rabbit IgG H&L AF488 secondary antibody. Part of the ValidAb™ range of highly validated, data-rich antibodies. |
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Validation data
Figure 1. IBA1 and Parvalbumin staining in rat brain using HB9317 Goat Anti-Rabbit H&L (AF488) and HB7393 Goat Anti-Mouse H&L (Janelia Fluor® 646)
Rat brain stained for microglia (HB7847) and parvalbumin expressing neurons (HB6457) using HB9317 goat anti-rabbit IgG H&L (AF488) and HB7393 goat anti-mouse H&L (Janelia Fluor® 646) 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 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB7847 (1:1,000 dilution) and HB6457 (1:1,000 dilution, 1µg/ml). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) and HB7393 goat anti-mouse H&L (Janelia Fluor® 646) 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 as a z-stack (0.5µm spacing) using a Leica DMI6000B fluorescence microscope with a 40x objective using DAP (12ms exposure), L5 (35ms exposure) and Y5 (40ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Neurons stained for NeuN in the rat hippocampus using HB6498 and HB9317 Goat Anti-Rabbit H&L (AF488).
Neurons in the rat hippocampus visualised using HB6498 (rabbit polyclonal anti-NeuN) and HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (1:2,000 dilution). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 as a z-stack (4.3µm spacing) using a Leica DMI6000B fluorescence microscope with a 10x objective using DAP (35ms exposure) and L5 (100ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Neuronal projections stained for Neurofilament L (NfL) in rat brainstem using HB7266 and HB9317 Goat Anti-Rabbit H&L (AF488).
Axons in the brainstem of a rat visualised using HB7266 (rabbit monoclonal anti-NfL) and HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 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). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 as a z-stack (0.3µm spacing) using a Leica DMI6000B fluorescence microscope with a 40x objective using DAP (10ms exposure) and L5 (35ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Calretinin staining in rat cerebellum using HB6494 and HB9317 Goat Anti-Rabbit H&L (AF488).
Rat cerebellum stained for calretinin using HB6494 (rabbit polyclonal antibody) using HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6494 (1:2,000 dilution). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 (40ms exposure) and L5 (200ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. IBA1 staining in the hippocampal region of rat brain using HB7847 and HB9317 Goat Anti-Rabbit H&L (AF488)
Hippocampal microglia stained for IBA1 using HB7847 and HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB7847 (1:1,000 dilution). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 as a z-stack (0.5µm spacing) using a Leica DMI6000B fluorescence microscope with a 40x objective using DAP (3ms exposure) and L5 (29ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Neuronal projections stained for Neurofilament L (NfL) in rat cortex using HB7266 and HB9317 Goat Anti-Rabbit H&L (AF488).
Rat cortical neurons visualised using HB7266 (rabbit monoclonal anti-NfL) and HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 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). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 as a z-stack (0.3µm spacing) using a Leica DMI6000B fluorescence microscope with a 40x objective using DAP (6ms exposure) and L5 (35ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 7. Calretinin staining in rat cerebellum using HB6494 and HB9317 Goat Anti-Rabbit H&L (AF488).
Rat cerebellum stained for calretinin using HB6494 (rabbit polyclonal antibody) using HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6494 (1:2,000 dilution). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 (40ms exposure) and L5 (200ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 1. IBA1 and Parvalbumin staining in rat brain using HB9317 Goat Anti-Rabbit H&L (AF488) and HB7393 Goat Anti-Mouse H&L (Janelia Fluor® 646)
Rat brain stained for microglia (HB7847) and parvalbumin expressing neurons (HB6457) using HB9317 goat anti-rabbit IgG H&L (AF488) and HB7393 goat anti-mouse H&L (Janelia Fluor® 646) 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 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB7847 (1:1,000 dilution) and HB6457 (1:1,000 dilution, 1µg/ml). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) and HB7393 goat anti-mouse H&L (Janelia Fluor® 646) 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 as a z-stack (0.5µm spacing) using a Leica DMI6000B fluorescence microscope with a 40x objective using DAP (12ms exposure), L5 (35ms exposure) and Y5 (40ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Neurons stained for NeuN in the rat hippocampus using HB6498 and HB9317 Goat Anti-Rabbit H&L (AF488).
Neurons in the rat hippocampus visualised using HB6498 (rabbit polyclonal anti-NeuN) and HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (1:2,000 dilution). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 as a z-stack (4.3µm spacing) using a Leica DMI6000B fluorescence microscope with a 10x objective using DAP (35ms exposure) and L5 (100ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Neuronal projections stained for Neurofilament L (NfL) in rat brainstem using HB7266 and HB9317 Goat Anti-Rabbit H&L (AF488).
Axons in the brainstem of a rat visualised using HB7266 (rabbit monoclonal anti-NfL) and HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 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). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 as a z-stack (0.3µm spacing) using a Leica DMI6000B fluorescence microscope with a 40x objective using DAP (10ms exposure) and L5 (35ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Calretinin staining in rat cerebellum using HB6494 and HB9317 Goat Anti-Rabbit H&L (AF488).
Rat cerebellum stained for calretinin using HB6494 (rabbit polyclonal antibody) using HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6494 (1:2,000 dilution). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 (40ms exposure) and L5 (200ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. IBA1 staining in the hippocampal region of rat brain using HB7847 and HB9317 Goat Anti-Rabbit H&L (AF488)
Hippocampal microglia stained for IBA1 using HB7847 and HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB7847 (1:1,000 dilution). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 as a z-stack (0.5µm spacing) using a Leica DMI6000B fluorescence microscope with a 40x objective using DAP (3ms exposure) and L5 (29ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Neuronal projections stained for Neurofilament L (NfL) in rat cortex using HB7266 and HB9317 Goat Anti-Rabbit H&L (AF488).
Rat cortical neurons visualised using HB7266 (rabbit monoclonal anti-NfL) and HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 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). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 as a z-stack (0.3µm spacing) using a Leica DMI6000B fluorescence microscope with a 40x objective using DAP (6ms exposure) and L5 (35ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 7. Calretinin staining in rat cerebellum using HB6494 and HB9317 Goat Anti-Rabbit H&L (AF488).
Rat cerebellum stained for calretinin using HB6494 (rabbit polyclonal antibody) using HB9317 goat anti-rabbit IgG H&L (AF488) 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 15 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6494 (1:2,000 dilution). This was followed by a two-hour incubation with HB9317 goat anti-rabbit IgG H&L (AF488) secondary antibody 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 (40ms exposure) and L5 (200ms exposure) filters. Following capture, deconvolution was carried out using Huygens Professional before 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, 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 | 490 nm |
| Max emission wavelength | 525 nm |
| Emission color | Green |
| Closest laser lines | 488 nm |
| Spectrally similar dyes | Alexa Fluor® 488, Atto 488, DyLight® 488, FITC |
| 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 (AF488) preadsorbed ValidAb™
References are publications that support the biological activity of the product
-
Human Neutrophils Present Mild Activation by Zika Virus But Reduce the Infection of Susceptible Cells.
Aggio JB et al (2022) Frontiers in immunology 13 : 784443 -
The Role of Wnt/β-Catenin Pathway Mediators in Aortic Valve Stenosis.
Khan K et al (2020) Frontiers in cell and developmental biology 8 : 862
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