Anti-GFAP antibody ValidAbTM
(HB6406)
Product overview
| Name | Anti-GFAP antibody ValidAbTM |
| Host | Chicken |
| Clonality | Polyclonal |
| Target | GFAP |
| Description | Antibody to GFAP - cytoskeletal protein used as an astrocyte marker. Part of the ValidAb™ range of highly validated, data-rich antibodies. |
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Validation data
Figure 1. An astrocyte surrounded by neurons in a cultured rat neuron preparation.
HB6406 stains GFAP in the star shaped projections of an astrocyte surrounded by neurofilament L stained with HB7266 (rabbit monoclonal). Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6406 (chicken polyclonal anti-GFAP) was incubated overnight (4°C) at a 1:8,000 dilution with HB7266 (rabbit monoclonal anti-NfL) at a 1:4,000 dilution (0.25µg/ml). This was followed by a one hour incubation with secondary antibodies (Polyclonal donkey anti-chicken Alexa Fluor 488 conjugated, Thermofisher A78948, 1:300 dilution and polyclonal goat anti-rabbit DyLight 550 conjugated, Thermofisher SA510033, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualize cell nuclei. For more detail please see our ICC 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 Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (21.1% power, PMT: 553V gain), 488nm (0.1% power, Hyd: 10% gain) and 561nm (0.1% power, Hyd: 10% gain) laser lines in a z-stack (0.42µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Stained hippocampal CA1 showing astrocytes, neurons and parvalbumin positive interneurons.
A section of hippocampal CA1 stained by HB6406 for GFAP (chicken polyclonal), HB6457 for Parvalbumin (mouse monoclonal) and HB6498 for NeuN (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6457 (1:1000, 1µg/ml) and HB6498 (1:4000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 in Lightning deconvolution mode using a 40x objective (2.25x zoom), 405nm (17.8% power, PMT: 559V gain), 488nm (0.25% power, Hyd: 10% gain) and 633nm (5.0% power, Hyd: 40% gain) laser lines in a z-stack (1.9µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Whole hippocampal section showing astrocytes, neurons and parvalbumin positive interneurons.
Rat hippocampus stained by HB6406 for GFAP (chicken polyclonal), HB6457 for Parvalbumin (mouse monoclonal) and HB6498 for NeuN (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6457 (1:1000, 1µg/ml) and HB6498 (1:4000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 10x objective (DAPI: 26.3ms, L5: 22ms, RHO: 150ms, Y5: 500ms exposures) in a z-stack (4µm spacing). The image was 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).
Figure 4. Populations of astrocytes and neurons in a cultured rat neuron preparation.
HB6406 stains the GFAP positive glia that surround and support the neurofilament L expressing neurones (HB7266 stained: rabbit monoclonal) in a rat cultured neuron preparation. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6406 (chicken polyclonal anti-GFAP) was incubated overnight (4°C) at a 1:2,000 dilution with HB7266 (rabbit monoclonal anti-NfL) at a 1:1,000 dilution (1µg/ml). This was followed by a one hour incubation with secondary antibodies (Polyclonal donkey anti-chicken Alexa Fluor 488 conjugated, Thermofisher A78948, 1:300 dilution and polyclonal goat anti-rabbit DyLight 550 conjugated, Thermofisher SA510033, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC 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 as a tilescan in Lightning deconvolution mode using a 40x objective (2.25x zoom), 405nm (28.9% power, PMT: 569V gain), 488nm (0.05% power, Hyd: 10% gain) and 561nm (0.1% power, Hyd: 10% gain) laser lines in a z-stack (0.42µm 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 HB6406 staining in rat cortex and cerebellum.
HB6406 produces strong staining of astrocytes in cortex and cerebellum at dilutions as low at 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight in HB6406 dilutions ranging from 1:2,000 to 1:8,000. This was followed by a two hour incubation with polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher, A78948 secondary antibody. DAPI (HB0747) was used at 1µg/ml to visualize cell nuclei. 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 as a z-stack with a 20x objective before being deconvolved in Huygens professional software and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Exposure times were as follows:
|
Dilution |
Cortex |
Cerebellum |
||
|
DAPI |
GFP |
DAPI |
GFP |
|
|
1:2,000 |
50ms |
229.8ms |
40ms |
120.6ms |
|
1:4,000 |
50ms |
229.8ms |
50ms |
229.8ms |
|
1:6,000 |
95.6ms |
211.8ms |
50ms |
229.8ms |
Figure 6. Concentration response of HB6406 staining in cultured rat neurons.
HB6406 produces strong staining of astrocytes in a cultured neuron preparation at dilutions as low at 1:8,000. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6406 (chicken polyclonal anti-GFAP) was incubated overnight (4°C) at a dilutions ranging from 1:,1000 to 1:8,000. This was followed by a one hour incubation with a polyclonal donkey anti-chicken Alexa Fluor 488 conjugated secondary antibody (Thermofisher A78948, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera as a z-stack before being deconvolved in Huygens professional software and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Exposure times were as follows:
- 1:1,000: DAPI: 144.1ms, L5: 155.1ms. 10x objective
- 1:2,000: DAPI: 17.0ms, L5: 4ms. 20x objective
- 1:4,000: DAPI: 17.0ms, L5: 29ms. 20x objective
- 1:8,000: DAPI: 17.0ms, L5: 29ms. 20x objective
Figure 7. GFAP expression in various tissue lysates and preparations.
HB6406 revealed the ≈55kDa band associated with GFAP only in neural tissue samples. 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 12% acrylamide gel alongside a protein ladder (NEB Prestained protein standard, P7718S) before being run at 60V for 30 minutes followed by 130V for 100 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 HB6406 at a 1:8,000 dilution. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal donkey anti-chicken HRP conjugated, Thermofisher, SA1300) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 6 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 (Sigma Aldrich A3682) for 2hrs and visualized again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).
Figure 8. Concentration response of HB6406 staining in a rat brain cytosol preparation.
HB6406 shows strong affinity for GFAP with bands visible at as low as a 1 in 128,000 dilution. Method: cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Rat cytosol samples were loaded (20µg / lane) onto a 12% acrylamide gel alongside a protein ladder (NEB Prestained protein standard, P7718S) before being run at 60V for 30 minutes followed by 120V for 100 minutes. Wet transfer to a PVDF membrane was completed in 80 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6406. Each strip was incubated separately with a separate HB6406 concentration with this ranging from 1:1000 to 1:256,000 dilutions. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal donkey anti-chicken HRP conjugated, Thermofisher, SA1300) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 8 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.
Figure 9. Independent antibody validation of HB6406 and HB8001 in a cultured rat neuron preparation.
HB6406 and HB8001 staining completely overlaps in a cultured neuron preparation therefore showing strong evidence for specificity. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6406 (chicken polyclonal anti-GFAP) was incubated overnight (4°C) at a 1:2,000 dilution with HB8001 (rabbit polyclonal anti-GFAP) at a 1:2,000 dilution. This was followed by a one hour incubation with secondary antibodies (Polyclonal donkey anti-chicken Alexa Fluor 488 conjugated, Thermofisher A78948, 1:300 dilution and polyclonal goat anti-rabbit DyLight 550 conjugated, Thermofisher SA510033, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC 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 as a tilescan in Lightning deconvolution mode using a 40x objective (2.25x zoom), 405nm (21.1% power, PMT: 512V gain), 488nm (0.05% power, Hyd: 10% gain) and 561nm (0.1% power, Hyd: 10% gain) laser lines in a z-stack (0.39µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 10. Populations of astrocytes and neurones in a cultured rat neuron preparation.
HB6406 stains the GFAP positive glia that surround and support the neurofilament L expressing neurones (HB7266 stained: rabbit monoclonal) in a rat cultured neuron preparation. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6406 (chicken polyclonal anti-GFAP) was incubated overnight (4°C) at a 1:2,000 dilution with HB7266 (rabbit monoclonal anti-NfL) at a 1:1,000 dilution (1µg/ml). This was followed by a one hour incubation with secondary antibodies (Polyclonal donkey anti-chicken Alexa Fluor 488 conjugated, Thermofisher A78948, 1:300 dilution and polyclonal goat anti-rabbit DyLight 550 conjugated, Thermofisher SA510033, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 14.6ms, L5: 14.6ms, RHO, 33.7ms exposures) in a z-stack (0.59µm spacing). The image was 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).
Figure 11. Astrocytes stained for GFAP with HB6406 in the cerebellum
Rat cerebellum stained by HB6406 for GFAP. 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 HB6406 (1:2000 dilution). This was followed by a two hour incubation with polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948 secondary antibody. 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 10x objective (DAPI: 40ms, GFP: 120.6ms exposures) in a z-stack (2.4µm spacing). The image was 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).
Figure 1. An astrocyte surrounded by neurons in a cultured rat neuron preparation.
HB6406 stains GFAP in the star shaped projections of an astrocyte surrounded by neurofilament L stained with HB7266 (rabbit monoclonal). Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6406 (chicken polyclonal anti-GFAP) was incubated overnight (4°C) at a 1:8,000 dilution with HB7266 (rabbit monoclonal anti-NfL) at a 1:4,000 dilution (0.25µg/ml). This was followed by a one hour incubation with secondary antibodies (Polyclonal donkey anti-chicken Alexa Fluor 488 conjugated, Thermofisher A78948, 1:300 dilution and polyclonal goat anti-rabbit DyLight 550 conjugated, Thermofisher SA510033, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualize cell nuclei. For more detail please see our ICC 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 Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (21.1% power, PMT: 553V gain), 488nm (0.1% power, Hyd: 10% gain) and 561nm (0.1% power, Hyd: 10% gain) laser lines in a z-stack (0.42µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. Stained hippocampal CA1 showing astrocytes, neurons and parvalbumin positive interneurons.
A section of hippocampal CA1 stained by HB6406 for GFAP (chicken polyclonal), HB6457 for Parvalbumin (mouse monoclonal) and HB6498 for NeuN (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6457 (1:1000, 1µg/ml) and HB6498 (1:4000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 in Lightning deconvolution mode using a 40x objective (2.25x zoom), 405nm (17.8% power, PMT: 559V gain), 488nm (0.25% power, Hyd: 10% gain) and 633nm (5.0% power, Hyd: 40% gain) laser lines in a z-stack (1.9µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Whole hippocampal section showing astrocytes, neurons and parvalbumin positive interneurons.
Rat hippocampus stained by HB6406 for GFAP (chicken polyclonal), HB6457 for Parvalbumin (mouse monoclonal) and HB6498 for NeuN (rabbit polyclonal). 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 HB6406 (1:2000 dilution), HB6457 (1:1000, 1µg/ml) and HB6498 (1:4000 dilution, 0.25µg/ml). This was followed by a two hour incubation with secondary antibodies at a 1:300 dilution (polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948; polyclonal goat anti-mouse Janelia Fluor 549; Goat anti-rabbit DyLight 650, Thermofisher 11804574). 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 10x objective (DAPI: 26.3ms, L5: 22ms, RHO: 150ms, Y5: 500ms exposures) in a z-stack (4µm spacing). The image was 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).
Figure 4. Populations of astrocytes and neurons in a cultured rat neuron preparation.
HB6406 stains the GFAP positive glia that surround and support the neurofilament L expressing neurones (HB7266 stained: rabbit monoclonal) in a rat cultured neuron preparation. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6406 (chicken polyclonal anti-GFAP) was incubated overnight (4°C) at a 1:2,000 dilution with HB7266 (rabbit monoclonal anti-NfL) at a 1:1,000 dilution (1µg/ml). This was followed by a one hour incubation with secondary antibodies (Polyclonal donkey anti-chicken Alexa Fluor 488 conjugated, Thermofisher A78948, 1:300 dilution and polyclonal goat anti-rabbit DyLight 550 conjugated, Thermofisher SA510033, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC 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 as a tilescan in Lightning deconvolution mode using a 40x objective (2.25x zoom), 405nm (28.9% power, PMT: 569V gain), 488nm (0.05% power, Hyd: 10% gain) and 561nm (0.1% power, Hyd: 10% gain) laser lines in a z-stack (0.42µm 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 HB6406 staining in rat cortex and cerebellum.
HB6406 produces strong staining of astrocytes in cortex and cerebellum at dilutions as low at 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 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight in HB6406 dilutions ranging from 1:2,000 to 1:8,000. This was followed by a two hour incubation with polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher, A78948 secondary antibody. DAPI (HB0747) was used at 1µg/ml to visualize cell nuclei. 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 as a z-stack with a 20x objective before being deconvolved in Huygens professional software and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Exposure times were as follows:
|
Dilution |
Cortex |
Cerebellum |
||
|
DAPI |
GFP |
DAPI |
GFP |
|
|
1:2,000 |
50ms |
229.8ms |
40ms |
120.6ms |
|
1:4,000 |
50ms |
229.8ms |
50ms |
229.8ms |
|
1:6,000 |
95.6ms |
211.8ms |
50ms |
229.8ms |
Figure 6. Concentration response of HB6406 staining in cultured rat neurons.
HB6406 produces strong staining of astrocytes in a cultured neuron preparation at dilutions as low at 1:8,000. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6406 (chicken polyclonal anti-GFAP) was incubated overnight (4°C) at a dilutions ranging from 1:,1000 to 1:8,000. This was followed by a one hour incubation with a polyclonal donkey anti-chicken Alexa Fluor 488 conjugated secondary antibody (Thermofisher A78948, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera as a z-stack before being deconvolved in Huygens professional software and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Exposure times were as follows:
- 1:1,000: DAPI: 144.1ms, L5: 155.1ms. 10x objective
- 1:2,000: DAPI: 17.0ms, L5: 4ms. 20x objective
- 1:4,000: DAPI: 17.0ms, L5: 29ms. 20x objective
- 1:8,000: DAPI: 17.0ms, L5: 29ms. 20x objective
Figure 7. GFAP expression in various tissue lysates and preparations.
HB6406 revealed the ≈55kDa band associated with GFAP only in neural tissue samples. 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 12% acrylamide gel alongside a protein ladder (NEB Prestained protein standard, P7718S) before being run at 60V for 30 minutes followed by 130V for 100 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 HB6406 at a 1:8,000 dilution. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal donkey anti-chicken HRP conjugated, Thermofisher, SA1300) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 6 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 (Sigma Aldrich A3682) for 2hrs and visualized again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).
Figure 8. Concentration response of HB6406 staining in a rat brain cytosol preparation.
HB6406 shows strong affinity for GFAP with bands visible at as low as a 1 in 128,000 dilution. Method: cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Rat cytosol samples were loaded (20µg / lane) onto a 12% acrylamide gel alongside a protein ladder (NEB Prestained protein standard, P7718S) before being run at 60V for 30 minutes followed by 120V for 100 minutes. Wet transfer to a PVDF membrane was completed in 80 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6406. Each strip was incubated separately with a separate HB6406 concentration with this ranging from 1:1000 to 1:256,000 dilutions. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal donkey anti-chicken HRP conjugated, Thermofisher, SA1300) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 8 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.
Figure 9. Independent antibody validation of HB6406 and HB8001 in a cultured rat neuron preparation.
HB6406 and HB8001 staining completely overlaps in a cultured neuron preparation therefore showing strong evidence for specificity. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6406 (chicken polyclonal anti-GFAP) was incubated overnight (4°C) at a 1:2,000 dilution with HB8001 (rabbit polyclonal anti-GFAP) at a 1:2,000 dilution. This was followed by a one hour incubation with secondary antibodies (Polyclonal donkey anti-chicken Alexa Fluor 488 conjugated, Thermofisher A78948, 1:300 dilution and polyclonal goat anti-rabbit DyLight 550 conjugated, Thermofisher SA510033, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC 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 as a tilescan in Lightning deconvolution mode using a 40x objective (2.25x zoom), 405nm (21.1% power, PMT: 512V gain), 488nm (0.05% power, Hyd: 10% gain) and 561nm (0.1% power, Hyd: 10% gain) laser lines in a z-stack (0.39µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 10. Populations of astrocytes and neurones in a cultured rat neuron preparation.
HB6406 stains the GFAP positive glia that surround and support the neurofilament L expressing neurones (HB7266 stained: rabbit monoclonal) in a rat cultured neuron preparation. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6406 (chicken polyclonal anti-GFAP) was incubated overnight (4°C) at a 1:2,000 dilution with HB7266 (rabbit monoclonal anti-NfL) at a 1:1,000 dilution (1µg/ml). This was followed by a one hour incubation with secondary antibodies (Polyclonal donkey anti-chicken Alexa Fluor 488 conjugated, Thermofisher A78948, 1:300 dilution and polyclonal goat anti-rabbit DyLight 550 conjugated, Thermofisher SA510033, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 14.6ms, L5: 14.6ms, RHO, 33.7ms exposures) in a z-stack (0.59µm spacing). The image was 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).
Figure 11. Astrocytes stained for GFAP with HB6406 in the cerebellum
Rat cerebellum stained by HB6406 for GFAP. 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 HB6406 (1:2000 dilution). This was followed by a two hour incubation with polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948 secondary antibody. 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 connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 10x objective (DAPI: 40ms, GFP: 120.6ms exposures) in a z-stack (2.4µm spacing). The image was 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).
Product information
| Immunogen | Recombinant human GFAP (isoform 1) expressed in and purified from E. coli |
| Isotype | IgY |
| Purification | Unpurified |
| Formulation | Lyophilised. When reconstituted contains IgY preparation with 5mM sodium azide and 1% recombinant BSA. |
| Predicted species reactivity | Mouse, Rat, Human, Pig, Horse, Cow |
| Tested species reactivity | Mouse, Rat |
Tested applications
| Applications | ICC, WB, IHC(IF) |
| Western blot optimal concentration | 1:8,000 dilution as tested in a rat brain cytosol preparation. |
| IHC(IF) optimal concentration | 1:4,000 dilution as tested in free-floating paraformaldehyde fixed horiztonal rat brain sections. |
| ICC optimal concentration | 1:8,000 dilution as tested in cultured rat neurones. |
| Positive control | GFAP is highly expressed in neural tissues containing astrocytes. It is not widely expressed in cell lines, however it is in specific lines such as U-87 MG. |
| Negative control | Most non-neural tissues. |
| Open data link | Please follow this link to OSF |
Target information
| UniProt ID | P14136 |
| Structure image |  |
| Gene name | GFAP |
| NCBI full gene name | glial fibrillary acidic protein |
| Entrez gene ID |
| Amino acids | 432 (49.9kDa) |
| Isoforms | GFAP has three confirmed and 21 potential isoforms. Isoform 1 (GFAP alpha): canonical, 49.9kDa; Isoform 2 (GFAP epsilon): amino acid changes between positions 391 and 432, 49.5kDa; Isoform 3 (GFAP kappa): amino acid changes between positions 391 and 432, 50.3kDa |
| Expression | GFAP is primarily expressed within astrocytes of the central nervous system alongside also expressing in non-myelinating Schwann cells of the peripheral nervous system and satellite cells of the peripheral ganglia. GFAP expression has also been reported in Leydig cells of the testis alongside stellate cells from the pancreas and liver in rats. |
| Subcellular expression | GFAP is a key cytoskeletal component therefore is widely expressed as bundles of GFAP positive fibres. |
| Processing | Following translation, no processing is required for GFAP to reach its active conformation. |
| Post translational modifications | GFAP is subjected to numerous post-translational modifications including 9 phosphorylation sites which are the target of AURKB and ROCK1 alongside 5 separate citrullination sites. |
| Homology (compared to human) | Rat, mouse and human GFAP proteins have a 90% similarity score in a direct BLAST comparison. |
| Similar proteins | Other type III intermediate filament proteins have homology with GFAP including Vimentin (58%), Desmin (59%) and Peripherin (56%) when assessed using BLAST. |
Storage & Handling
| Storage instructions | -20°C then use reconstitution advice |
| Handling | Upon receipt store at either -20°C or -80°C. When ready to use there are three options:
For more information read our guide on the best care for your product. 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. |
| 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-GFAP antibody ValidAbTM
References are publications that support the biological activity of the product
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Importance of GFAP isoform-specific analyses in astrocytoma.
van Bodegraven EJ et al (2019) Glia 67 : 1417-1433 -
The role of GFAP and vimentin in learning and memory.
Wilhelmsson U et al (2019) Biological chemistry 400 : 1147-1156 -
GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain.
Garcia AD et al (2004) Nature neuroscience 7 : 1233-41 -
Glial fibrillary acidic protein: GFAP-thirty-one years (1969-2000).
Eng LF et al (2000) Neurochemical research 25 : 1439-51 -
GFAP and astrogliosis.
Eng LF et al (1994) Brain pathology (Zurich, Switzerland) 4 : 229-37
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