Anti-IBA1 antibody ValidAbTM
(HB7847)
Product overview
| Name | Anti-IBA1 antibody ValidAbTM |
| Host | Rabbit |
| Clonality | Polyclonal |
| Target | IBA1 |
| Description | Antibody to IBA1 - calcium binding protein widely used as a marker for microglial cells. Part of the ValidAb™ range of highly validated, data-rich antibodies. |
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Validation data
Figure 1. Cerebellar microglia stained for IBA1 with HB7847.
Rat cerebellum showing microglia stained for IBA1 with HB7847 and Purkinje cells stained for βIII-tubulin with HB6639. 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 (1:2,000 dilution) and HB6639 (1:2,000 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 550, Thermofisher, 84540 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). 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 in as a tilescan in Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (16.0% power, PMT: 681V gain), 561nm (4% power, Hyd: 10% gain) and 633nm(4% power, Hyd: 15.3% gain) laser lines in a z-stack (0.96µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Cortical microglia stained for IBA1 with HB7847
Rat cortex showing microglia stained for IBA1 with HB7847. 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 (1:2,000 dilution). This was followed by a two-hour incubation with secondary antibody at a 1:300 dilution (polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured as a tilescan using a 40x objective in a z-stack (0.3µm spacing). The image was captured using DAP (10ms exposure) and Y5 (39ms exposure) filters. The stack was 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. Microglia and neurons stained in the caudate putamen
Rat CPu showing microglia stained for IBA1 with HB7847 and neurons stained for βIII-tubulin with HB6639. 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 (1:4,000 dilution) and HB6639 (1:4,000 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 550, Thermofisher, 84540 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 20x objective in a z-stack (0.59µm spacing). The image was captured using DAP (25ms exposure), RHO (100ms exposure) and Y5 (150ms exposure) filters. The stack was 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. IBA1 expressing microglia in the cortex
Rat cortex showing microglia stained for IBA1 with HB7847 and neurons stained for βIII-tubulin with HB6639. 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 (1:2,000 dilution) and HB6639 (1:2,000 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 550, Thermofisher, 84540 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured as a tilescan using a 40x objective in a z-stack (0.3µm spacing). The image was captured using DAP (10ms exposure), RHO (33ms exposure) and Y5 (39ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Concentration response of HB7847 staining in rat brain.
HB7847 labels microglia with high signal to noise down to a dilution of 1:8,000. 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 to 1:8,000 dilutions). This was followed by a two-hour incubation with secondary antibody at a 1:300 dilution (polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. Images were captured using a 20x objective in a z-stack. Images were deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682). Exposure times were as follows:
• 1:1000: DAPI: 24.5ms, Y5: 39ms
• 1:2000: DAPI: 20ms, Y5: 87ms
• 1:4000: DAPI: 25ms, Y5: 150ms
• 1:8000: DAPI: 20ms, Y5: 161ms
Figure 6. Independent antibody validation of HB6589 in rat cortex
The staining patterns of HB6589 and a competitor chicken polyclonal anti-IBA1 antibody in rat cortex overlap showing strong evidence for specificity. 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 (1:2,000 dilution) and competitor polyclonal chicken anti-IBA1 antibody (1:500 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken DyLight 488, Thermofisher, A78948 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 20x objective in a z-stack (1µm spacing). The image was captured using DAP (20ms exposure), L5 (103ms exposure) and Y5 (226ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 7. Microglia expressing IBA1 in rat brain
Rat brain showing microglia stained for IBA1 with HB7847. 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 secondary antibody at a 1:300 dilution (polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured as a tilescan using a 40x objective in a z-stack (0.3µm spacing). The image was captured using DAP (9ms exposure) and Y5 (39ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 8. Microglia and Purkinje cells in the cerebellum
Rat cerebellum showing microglia stained for IBA1 with HB7847 and Purkinje cells stained for βIII-tubulin with HB6639. 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 (1:2,000 dilution) and HB6639 (1:2,000 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 550, Thermofisher, 84540 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 20x objective in a z-stack (0.59µm spacing). The image was captured using DAP (20ms exposure), RHO (80ms exposure) and Y5 (87ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 9. Microglia surrounded by dopaminergic projections in the striatum
Rat caudate putamen showing microglia stained for IBA1 with HB7847 and dopaminergic terminals stained for tyrosine hydroxylase with HB6589. 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 HB6589 (1:1,000 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken DyLight 488, Thermofisher, A78948 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured as a tilescan using a 40x objective in a z-stack (0.3µm spacing). The image was captured using DAP (9ms exposure), RHO (142ms exposure) and Y5 (39ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 10. Independent antibody validation of HB6589 in rat CA1
The staining patterns of HB6589 and a competitor chicken polyclonal anti-IBA1 antibody in rat CA1 overlap showing strong evidence for specificity. 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 (1:2,000 dilution) and competitor polyclonal chicken anti-IBA1 antibody (1:500 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken DyLight 488, Thermofisher, A78948 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 20x objective in a z-stack (1µm spacing). The image was captured using DAP (20ms exposure), L5 (103ms exposure) and Y5 (226ms exposure) filters. The stack was 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. Cerebellar microglia stained for IBA1 with HB7847.
Rat cerebellum showing microglia stained for IBA1 with HB7847 and Purkinje cells stained for βIII-tubulin with HB6639. 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 (1:2,000 dilution) and HB6639 (1:2,000 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 550, Thermofisher, 84540 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). 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 in as a tilescan in Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (16.0% power, PMT: 681V gain), 561nm (4% power, Hyd: 10% gain) and 633nm(4% power, Hyd: 15.3% gain) laser lines in a z-stack (0.96µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Cortical microglia stained for IBA1 with HB7847
Rat cortex showing microglia stained for IBA1 with HB7847. 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 (1:2,000 dilution). This was followed by a two-hour incubation with secondary antibody at a 1:300 dilution (polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured as a tilescan using a 40x objective in a z-stack (0.3µm spacing). The image was captured using DAP (10ms exposure) and Y5 (39ms exposure) filters. The stack was 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. Microglia and neurons stained in the caudate putamen
Rat CPu showing microglia stained for IBA1 with HB7847 and neurons stained for βIII-tubulin with HB6639. 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 (1:4,000 dilution) and HB6639 (1:4,000 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 550, Thermofisher, 84540 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 20x objective in a z-stack (0.59µm spacing). The image was captured using DAP (25ms exposure), RHO (100ms exposure) and Y5 (150ms exposure) filters. The stack was 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. IBA1 expressing microglia in the cortex
Rat cortex showing microglia stained for IBA1 with HB7847 and neurons stained for βIII-tubulin with HB6639. 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 (1:2,000 dilution) and HB6639 (1:2,000 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 550, Thermofisher, 84540 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured as a tilescan using a 40x objective in a z-stack (0.3µm spacing). The image was captured using DAP (10ms exposure), RHO (33ms exposure) and Y5 (39ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 5. Concentration response of HB7847 staining in rat brain.
HB7847 labels microglia with high signal to noise down to a dilution of 1:8,000. 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 to 1:8,000 dilutions). This was followed by a two-hour incubation with secondary antibody at a 1:300 dilution (polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. Images were captured using a 20x objective in a z-stack. Images were deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682). Exposure times were as follows:
• 1:1000: DAPI: 24.5ms, Y5: 39ms
• 1:2000: DAPI: 20ms, Y5: 87ms
• 1:4000: DAPI: 25ms, Y5: 150ms
• 1:8000: DAPI: 20ms, Y5: 161ms
Figure 6. Independent antibody validation of HB6589 in rat cortex
The staining patterns of HB6589 and a competitor chicken polyclonal anti-IBA1 antibody in rat cortex overlap showing strong evidence for specificity. 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 (1:2,000 dilution) and competitor polyclonal chicken anti-IBA1 antibody (1:500 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken DyLight 488, Thermofisher, A78948 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 20x objective in a z-stack (1µm spacing). The image was captured using DAP (20ms exposure), L5 (103ms exposure) and Y5 (226ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 7. Microglia expressing IBA1 in rat brain
Rat brain showing microglia stained for IBA1 with HB7847. 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 secondary antibody at a 1:300 dilution (polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured as a tilescan using a 40x objective in a z-stack (0.3µm spacing). The image was captured using DAP (9ms exposure) and Y5 (39ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 8. Microglia and Purkinje cells in the cerebellum
Rat cerebellum showing microglia stained for IBA1 with HB7847 and Purkinje cells stained for βIII-tubulin with HB6639. 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 (1:2,000 dilution) and HB6639 (1:2,000 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal goat anti-mouse DyLight 550, Thermofisher, 84540 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 20x objective in a z-stack (0.59µm spacing). The image was captured using DAP (20ms exposure), RHO (80ms exposure) and Y5 (87ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 9. Microglia surrounded by dopaminergic projections in the striatum
Rat caudate putamen showing microglia stained for IBA1 with HB7847 and dopaminergic terminals stained for tyrosine hydroxylase with HB6589. 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 HB6589 (1:1,000 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken DyLight 488, Thermofisher, A78948 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured as a tilescan using a 40x objective in a z-stack (0.3µm spacing). The image was captured using DAP (9ms exposure), RHO (142ms exposure) and Y5 (39ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 10. Independent antibody validation of HB6589 in rat CA1
The staining patterns of HB6589 and a competitor chicken polyclonal anti-IBA1 antibody in rat CA1 overlap showing strong evidence for specificity. 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 (1:2,000 dilution) and competitor polyclonal chicken anti-IBA1 antibody (1:500 dilution). This was followed by a two-hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken DyLight 488, Thermofisher, A78948 and polyclonal goat anti-rabbit DyLight 650, Thermofisher, 11804574). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei and sections were mounted using MightyMount Antifade Fluorescence Mounting Medium (hardset). For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope. The image was captured using a 20x objective in a z-stack (1µm spacing). The image was captured using DAP (20ms exposure), L5 (103ms exposure) and Y5 (226ms exposure) filters. The stack was deconvolved using Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Product information
| Immunogen | C-terminal peptide of human IBA1 coupled to keyhole limpet haemocyanin (KLH) |
| Purification | Unpurified |
| Formulation | Lyophilised. When reconstituted contains serum with 1% recombinant BSA and 5mM sodium azide |
| Predicted species reactivity | Mouse, Rat, Human |
| Tested species reactivity | Mouse, Rat |
Tested applications
| Applications | IHC(IF) |
| IHC(IF) optimal concentration | 1:2,000 dilution as tested in free-floating paraformaldehyde fixed rat brain sections |
| Positive control | IBA1 is widely expressed in microglia across the CNS making brain tissue an excellent positive control. Adittionally there is high IBA1 expression in the spleen which is another popular positive control. |
| Negative control | IBA1 expression is absent in many popular cell lines such as HEK293T and HeLa making them a good negative control. |
| Open data link | Please follow this link to the OSF. |
Target information
| Other names | AIF1, Allograft inflammatory factor 1, ionized calcium-binding adapter molecule 1 |
| UniProt ID | P55008 |
| Structure image |  |
| Gene name | AIF1 |
| NCBI full gene name | Allograft inflammatory factor 1 |
| Entrez gene ID |
| Amino acids | 147 (16.7kDa) |
| Isoforms | IBA1 has three known isoforms:
|
| Expression | Expressed in myeloid lineage cells including microglia within the CNS and circulating macrophages. IBA1 is also expressed in dendritic cells and osteoclasts. |
| Subcellular expression | Cytosolic |
| Processing | IBA1 has the initiator methionine removed to form an active conformation |
| Post translational modifications | IBA1 is subject to phosphorylation on residues S2, S38 and S39 in adittion to acetylation on K11. |
| Homology (compared to human) | A BLAST search revealed the following homologies:
|
| Similar proteins | A BLAST search identified the following similar proteins to IBA1:
|
Storage & Handling
| Storage instructions | -20°C then use reconstitution advice |
| Reconstitution advice | We recommend reconstituting with either:
Take care when opening as the precipitate is extremely light and can easily be lost if disturbed. When reconstituting make sure that the antibody is thoroughly dissolved by pipetting up and down before giving the antibody a brief spin at <10,000g to make sure that all material is recovered and at the bottom of the tube. For more information please see our detailed guide on storing and using your antibody |
| 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) References for Anti-IBA1 antibody ValidAbTM
References are publications that support the biological activity of the product
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AIF1: Function and Connection with Inflammatory Diseases.
De Leon-Oliva D et al (2023) Biology 12 : -
Altered synaptic connectivity and brain function in mice lacking microglial adapter protein Iba1.
Lituma PJ et al (2021) Proceedings of the National Academy of Sciences of the United States of America 118 : -
Iba1 is an actin-cross-linking protein in macrophages/microglia.
Sasaki Y et al (2001) Biochemical and biophysical research communications 286 : 292-7 -
Microglia-specific localisation of a novel calcium binding protein, Iba1.
Ito D et al (1998) Brain research. Molecular brain research 57 : 1-9
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