Figure 1. GFAP expression in a rat cultured neurone preparation visualised using HB8267.
GFAP expression in the dense intermediate filament network of glial cells was successfully visualised using HB8267 at a 1:1000 dilution (1µg/ml). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 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 SP5-II confocal laser scanning microscope coupled to a Leica DMI 6000 inverted epifluorescence microscope. The image was captured using a 63x objective (2x zoom), 405nm (20.0% power) and 488nm (20.0% power) laser lines in a z-stack (0.335 µm spacing). Deconvolution was carried out using Huygens Essential (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. GFAP and Vimentin expression in a rat cultured neuron preparation visualised using HB8267 and an anti-Vimentin antibody.
GFAP and Vimentin are both expressed in glial cells and can be seen to express at different levels in individual cells creating a colour gradient across the cell population. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) with a goat anti-Vimentin antibody being incubated at a 1:500 dilution (2µg/ml). Secondary antibodies (Polyclonal donkey anti-mouse DyLight 488 conjugated, Thermofisher SA5-10166, 1:300 dilution and Polyclonal donkey anti-goat DyLight 594 conjugated, Thermofisher SA5-10088, 1:300 dilution) were incubated for 1 hour at room temperature. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured in a tile scan and z-stack (0.49µm spacing) using Lightning adaptive deconvolution and a 63x objective. Lasers used were 405nm (20.0% power, PMT: 712.7V gain), 496nm (1.0% power, HyD: 14.1% gain) and 561nm (1.5% power, HyD: 10% gain). The tile scan was merged using LASX before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. GFAP labelled astrocyte in rat brain.
HB8267 staining reveals an astrocyte in rat brain tissue surrounded by axonal processes labelled by HB7266 (rabbit monoclonal anti-neurofilament L). 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 transverse slices before sections were incubated in 1% NaBH4 for 30 minutes followed by 0.05M glycine for 30 minutes. Sections were blocked in 2% BSA, 3% goat serum before incubation overnight in HB8267 (1:1000 dilution, 1µg/ml) and HB7266 (1:2000 dilution, 0.5µg/ml). This was followed by a two hour incubation with secondary antibodies (polyclonal goat anti-rabbit DyLight 594 conjugated, Thermofisher 35561, 1:300 dilution and polyclonal goat anti-mouse Janelia Fluor 525 (HB8455) conjugated (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 SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 40x objective and 405nm (21.1% power, PMT: 592V gain), 561nm (0.5% power, HyD: 22.7% gain) and 514nm (1.0% power, HyD: 10.0% gain) lasers. Images were captured as a stack (0.49µm z-spacing) being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. GFAP expression in various tissue lysates and preparations.
HB8267 revealed a band of size 49kDa only present in brain derived 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 10% acrylamide gel alongside a protein ladder (Thermofisher, 26617) before being run at 60V for 40 minutes followed by 120V for 90 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 HB8267 at a 1:1000 dilution (1µg/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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: 10 min exposure, 700nm channel: 2 min exposure).
Figure 5. The effect of varying HB8267 concentration upon staining in rat hippocampus.
HB8267 produced a strong signal to noise ratio at dilutions as low as 1in4000 (250 ng/ml). Method: hippocampi were dissected from rat brains and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) for another 24hrs. A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 0.05M glycine for 30 minutes. Sections were blocked in 1% BSA, 22.52mg/ml glycine before incubation overnight in varying dilutions of HB8267 ranging from 1:500 (2µg/ml) to 1:4000 (0.25µg/ml) with a no primary step omitting the addition of HB8267 to the incubation buffer. This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). 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 DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI and I3 filters. Exposure times were as follows:
1:500 – DAPI 10x gain, 11ms exposure, I3 10x gain, 26.9ms exposure
1:1000 – DAPI 10x gain, 18.3ms exposure, I3 10x gain, 18.3ms exposure
1:2000 – DAPI 10x gain, 11.1ms exposure, I3 10x gain, 27.1ms exposure
1:4000 – DAPI 10x gain, 11.1ms exposure, I3 10x gain, 32.5ms exposure
No primary – DAPI: 10.18ms exposure, I3: 112.ms exposure (taken using different microscope: Leica DMI6000B with Photometric-Prime95B camera).
Images were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool before stacking and montage creation.
Figure 6. Concentration response of HB8267 staining in a rat brain cytosol preparation.
HB6429 shows consistent results with low background at dilutions as low as 1:16,000 (62.5 ng/ml). Method: Rat brain cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Samples were loaded (20µg / lane) onto a 12% acrylamide gel alongside a protein ladder (Thermofisher, 26616) before being run at 60V for 30 minutes followed by 120V for 100 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6429. Each strip was incubated separately with a separate HB6429 concentration with this ranging from 2µg/ml (1:500 dilution) to 31.25ng/ml (1:32,000 dilution). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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: 10 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 7. GFAP expression in a rat cultured neurone preparation visualised using HB8267.
GFAP expression in the dense intermediate filament network of glial cells was successfully visualised using HB8267 at a 1:1000 dilution (1µg/ml). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI LP (3.4x gain, 47.6ms exposure) and I3 (3.4x gain, 136.7ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 8. GFAP expression in various tissue lysates and preparations with GAPDH loading control.
HB8267 revealed a band of size 47kDa only present in brain derived 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 (Thermofisher, 26617) before being run at 60V for 30 minutes followed by 120V 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 HB8267 at a 1:1000 dilution (1µg/ml) and HB9177 (anti-GAPDH monoclonal antibody) at a 1:2,000 dilution (0.5µg/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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: 10 min exposure, 700nm channel: 2 min exposure).
Figure 9. GFAP expression in a rat cultured neurone preparation visualised using HB8267.
GFAP expression in the dense intermediate filament network of glial cells was successfully visualised using HB8267 at a 1:1000 dilution (1µg/ml). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI LP (5.9x gain, 46.2ms exposure) and I3 (5.9x gain, 46.2ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 10. GFAP and actin expression in a rat cultured neurone preparation visualised using HB8267.
GFAP expression was visualised using HB8267 whereas actin was stained using FITC-Phalloidin (HB0814) in glial cells. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution) and FITC phalloidin (1:40 dilution, 183nM).DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope using a 63x objective. Exictation was through 405nm (43.5% power, gain: 624), 488nm (51.3% power, gain: 846) and 532nm (41.3% power, gain: 737) laser lines in a z-stack (0.18µm spacing). Deconvolution was carried out using Huygens Essential (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 11. GFAP expression in a rat cultured neurone preparation visualised using HB8267.
GFAP expression in the dense intermediate filament network of glial cells was successfully visualised using HB8267 at a 1:1000 dilution (1µg/ml). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI LP (3.4x gain, 54.3ms exposure) and I3 (3.4x gain, 102.3ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 12. Dense populations of astrocytes in rat brain visualised using HB8267
HB8267 staining reveals both the dense network and the heterogeneity of structure of astrocytes present in rat brain. 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 followed by 0.05M glycine for 30 minutes. Sections were blocked in 2% BSA, 3% goat serum before incubation overnight in HB8267 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, 1:300 dilution, Thermofisher, 35503). 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 DMi6000 inverted epifluorescence microscope with Leica DFC365FX monochrome digital camera. The image was captured in a z-stack (2.4µm spacing) with a 20x objective, A4 (349ms exposure @ 1x gain) and GFP (228ms exposure @ 1x gain) filters. Images were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool before stacking and montage creation.
Figure 1. GFAP expression in a rat cultured neurone preparation visualised using HB8267.
GFAP expression in the dense intermediate filament network of glial cells was successfully visualised using HB8267 at a 1:1000 dilution (1µg/ml). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 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 SP5-II confocal laser scanning microscope coupled to a Leica DMI 6000 inverted epifluorescence microscope. The image was captured using a 63x objective (2x zoom), 405nm (20.0% power) and 488nm (20.0% power) laser lines in a z-stack (0.335 µm spacing). Deconvolution was carried out using Huygens Essential (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. GFAP and Vimentin expression in a rat cultured neuron preparation visualised using HB8267 and an anti-Vimentin antibody.
GFAP and Vimentin are both expressed in glial cells and can be seen to express at different levels in individual cells creating a colour gradient across the cell population. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) with a goat anti-Vimentin antibody being incubated at a 1:500 dilution (2µg/ml). Secondary antibodies (Polyclonal donkey anti-mouse DyLight 488 conjugated, Thermofisher SA5-10166, 1:300 dilution and Polyclonal donkey anti-goat DyLight 594 conjugated, Thermofisher SA5-10088, 1:300 dilution) were incubated for 1 hour at room temperature. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured in a tile scan and z-stack (0.49µm spacing) using Lightning adaptive deconvolution and a 63x objective. Lasers used were 405nm (20.0% power, PMT: 712.7V gain), 496nm (1.0% power, HyD: 14.1% gain) and 561nm (1.5% power, HyD: 10% gain). The tile scan was merged using LASX before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. GFAP labelled astrocyte in rat brain.
HB8267 staining reveals an astrocyte in rat brain tissue surrounded by axonal processes labelled by HB7266 (rabbit monoclonal anti-neurofilament L). 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 transverse slices before sections were incubated in 1% NaBH4 for 30 minutes followed by 0.05M glycine for 30 minutes. Sections were blocked in 2% BSA, 3% goat serum before incubation overnight in HB8267 (1:1000 dilution, 1µg/ml) and HB7266 (1:2000 dilution, 0.5µg/ml). This was followed by a two hour incubation with secondary antibodies (polyclonal goat anti-rabbit DyLight 594 conjugated, Thermofisher 35561, 1:300 dilution and polyclonal goat anti-mouse Janelia Fluor 525 (HB8455) conjugated (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 SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 40x objective and 405nm (21.1% power, PMT: 592V gain), 561nm (0.5% power, HyD: 22.7% gain) and 514nm (1.0% power, HyD: 10.0% gain) lasers. Images were captured as a stack (0.49µm z-spacing) being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. GFAP expression in various tissue lysates and preparations.
HB8267 revealed a band of size 49kDa only present in brain derived 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 10% acrylamide gel alongside a protein ladder (Thermofisher, 26617) before being run at 60V for 40 minutes followed by 120V for 90 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 HB8267 at a 1:1000 dilution (1µg/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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: 10 min exposure, 700nm channel: 2 min exposure).
Figure 5. The effect of varying HB8267 concentration upon staining in rat hippocampus.
HB8267 produced a strong signal to noise ratio at dilutions as low as 1in4000 (250 ng/ml). Method: hippocampi were dissected from rat brains and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) for another 24hrs. A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 0.05M glycine for 30 minutes. Sections were blocked in 1% BSA, 22.52mg/ml glycine before incubation overnight in varying dilutions of HB8267 ranging from 1:500 (2µg/ml) to 1:4000 (0.25µg/ml) with a no primary step omitting the addition of HB8267 to the incubation buffer. This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). 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 DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI and I3 filters. Exposure times were as follows:
1:500 – DAPI 10x gain, 11ms exposure, I3 10x gain, 26.9ms exposure
1:1000 – DAPI 10x gain, 18.3ms exposure, I3 10x gain, 18.3ms exposure
1:2000 – DAPI 10x gain, 11.1ms exposure, I3 10x gain, 27.1ms exposure
1:4000 – DAPI 10x gain, 11.1ms exposure, I3 10x gain, 32.5ms exposure
No primary – DAPI: 10.18ms exposure, I3: 112.ms exposure (taken using different microscope: Leica DMI6000B with Photometric-Prime95B camera).
Images were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool before stacking and montage creation.
Figure 6. Concentration response of HB8267 staining in a rat brain cytosol preparation.
HB6429 shows consistent results with low background at dilutions as low as 1:16,000 (62.5 ng/ml). Method: Rat brain cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Samples were loaded (20µg / lane) onto a 12% acrylamide gel alongside a protein ladder (Thermofisher, 26616) before being run at 60V for 30 minutes followed by 120V for 100 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6429. Each strip was incubated separately with a separate HB6429 concentration with this ranging from 2µg/ml (1:500 dilution) to 31.25ng/ml (1:32,000 dilution). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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: 10 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 7. GFAP expression in a rat cultured neurone preparation visualised using HB8267.
GFAP expression in the dense intermediate filament network of glial cells was successfully visualised using HB8267 at a 1:1000 dilution (1µg/ml). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI LP (3.4x gain, 47.6ms exposure) and I3 (3.4x gain, 136.7ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 8. GFAP expression in various tissue lysates and preparations with GAPDH loading control.
HB8267 revealed a band of size 47kDa only present in brain derived 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 (Thermofisher, 26617) before being run at 60V for 30 minutes followed by 120V 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 HB8267 at a 1:1000 dilution (1µg/ml) and HB9177 (anti-GAPDH monoclonal antibody) at a 1:2,000 dilution (0.5µg/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) 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: 10 min exposure, 700nm channel: 2 min exposure).
Figure 9. GFAP expression in a rat cultured neurone preparation visualised using HB8267.
GFAP expression in the dense intermediate filament network of glial cells was successfully visualised using HB8267 at a 1:1000 dilution (1µg/ml). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI LP (5.9x gain, 46.2ms exposure) and I3 (5.9x gain, 46.2ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 10. GFAP and actin expression in a rat cultured neurone preparation visualised using HB8267.
GFAP expression was visualised using HB8267 whereas actin was stained using FITC-Phalloidin (HB0814) in glial cells. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution) and FITC phalloidin (1:40 dilution, 183nM).DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope using a 63x objective. Exictation was through 405nm (43.5% power, gain: 624), 488nm (51.3% power, gain: 846) and 532nm (41.3% power, gain: 737) laser lines in a z-stack (0.18µm spacing). Deconvolution was carried out using Huygens Essential (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 11. GFAP expression in a rat cultured neurone preparation visualised using HB8267.
GFAP expression in the dense intermediate filament network of glial cells was successfully visualised using HB8267 at a 1:1000 dilution (1µg/ml). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI LP (3.4x gain, 54.3ms exposure) and I3 (3.4x gain, 102.3ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.
Figure 12. Dense populations of astrocytes in rat brain visualised using HB8267
HB8267 staining reveals both the dense network and the heterogeneity of structure of astrocytes present in rat brain. 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 followed by 0.05M glycine for 30 minutes. Sections were blocked in 2% BSA, 3% goat serum before incubation overnight in HB8267 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, 1:300 dilution, Thermofisher, 35503). 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 DMi6000 inverted epifluorescence microscope with Leica DFC365FX monochrome digital camera. The image was captured in a z-stack (2.4µm spacing) with a 20x objective, A4 (349ms exposure @ 1x gain) and GFP (228ms exposure @ 1x gain) filters. Images were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool before stacking and montage creation.
Product information
Immunogen
GFAP purified from porcine spinal cord
Clone number
GA-5
Isotype
IgG1
Purification
Protein A affinity chromatography
Concentration
1mg/ml
Formulation
Lyophilised. When reconstituted contains PBS with 15mM sodium azide and 1% recombinant albumin
Predicted species reactivity
Human, Mouse, Rat, Pig
Tested species reactivity
Mouse, Rat
Tested applications
Applications
ICC, WB, IHC(IF)
Western blot optimal concentration
1µg/ml (1:1000) as measured in rat brain cytosol
IHC(IF) optimal concentration
1µg/ml (1:1000) as measured in free-floating fixed hippocampal sections
ICC optimal concentration
1µg/ml (1:1000) as measured in neuronal cell culture
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. Please note that GFAP expression has been reported in a subset of pancreatic and hepatic cells in rats and mice kidney cells. It is generally poorly expressed in common cell lines such as HeLa or HEK293.
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:
Reconstitute with 100μl dH2O and store at 4°C
Reconstitue with 50μl dH2O and 50μl glycerol then store at -20°C
Reconstitue with 100μl dH2O, aliquot then snap freeze and store at -80°C
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.
Shipping conditions
Shipped at ambient temperature in a lyophilised format
Important
This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use
What guarantee do you have that my GFAP antibody will perform as expected?
We guarantee that your GFAP antibody will work for the applications and species we list on the datasheet. If the antibody fails to perform as expected then we are happy to offer a 100% refund guarantee. For more details please see our guarantee policy.
Will my GFAP antibody work against species that have not been listed on the datasheet?
A species not being listed doesn’t mean that the GFAP antibody won’t work, just that we haven’t tested it. If you test one of our antibodies in a new species please let us know (positive or negative)!
What protocols are available for use with this GFAP antibody
We have made a comprehensive collection of protocols that we have used in our experiments to validate this GFAP antibody.
What counterstains do you recommend for use in ICC and IHC with this GFAP antibody?
We recommend using either DAPI or Hoechst 33342 to label cell nuclei. In some experiments it is also helpful to label actin filaments in the cytoskeleton using a Phalloidin conjugate such as FITC Phalloidin or Rhodamine Phalloidin-TRITC.
Western Blot Protocol (1 MB) 
