Anti-IBA1 antibody ValidAb^TM

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).

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).

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).

HB7847 revealed the ≈18.5kDa band associated with IBA1 in rat spleen and brain samples from mice and rats. Two different exposure levels are shown as IBA1 is expressed at much higher levels in spleen that in the brain. No IBA1 reactivity is seen in the HEK293T sample which acts as a negative control. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples were loaded (20µg / lane) onto a 4-20% acrylamide gel alongside a protein ladder before being run at 160V for 45 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7847 at a 1:1,000 dilution. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate and a Licor Odyssey Fc imaging system (ECL channel: 5 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing the membrane was incubated in a 1:10,000 dilution of a polyclonal goat anti-mouse HRP conjugated secondary antibody for 2hrs and visualised again using Clarity Western ECL substrate and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).

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).

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

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).

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).

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).

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).

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).