Anti-GFAP antibody [EPR1034Y] ab68428 is a rabbit monoclonal antibody that is used in GFAP western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests- Validated for multiplex IHC on the Leica BOND® MAX using Opal reagents; and for VCAM1 IHC using Leica BOND™ RX- Antibody clone EPR1034Y has been tried and trusted by researchers since 2008 and is cited in >180 publications- One antibody for all your GFAP staining, use in GFAP western blotting, IHC, immunofluorescence and flow cytometryNew 20 ul size available
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.21% BSA
Liquid
Monoclonal
mIHC | Flow Cyt (Intra) | IHC-P | ICC/IF | IP | WB | IHC-Fr | |
---|---|---|---|---|---|---|---|
Human | Tested | Tested | Tested | Expected | Expected | Tested | Expected |
Mouse | Tested | Tested | Tested | Tested | Expected | Tested | Tested |
Rat | Tested | Expected | Expected | Tested | Tested | Tested | Tested |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/500 - 1/2000 | Notes - |
Species Mouse | Dilution info 1/500 - 1/2000 | Notes - |
Species Rat | Dilution info 1/500 - 1/2000 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/500 | Notes - |
Species Mouse | Dilution info 1/500 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/250 - 1/500 | Notes Perform heat-mediated antigen retrieval before commencing with IHC staining protocol. |
Species Human | Dilution info 1/250 - 1/500 | Notes Perform heat-mediated antigen retrieval before commencing with IHC staining protocol. |
Species | Dilution info | Notes |
---|---|---|
Species Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 0.1-1 µg/mL | Notes - |
Species Rat | Dilution info 0.1-1 µg/mL | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Rat | Dilution info 1/20 - 1/40 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human, Mouse | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/10000 | Notes For unpurified use at 1/50 000 - 1/100 000. |
Species Rat | Dilution info 1/10000 | Notes For unpurified use at 1/50 000 - 1/100 000. |
Species Human | Dilution info 1/10000 | Notes For unpurified use at 1/50 000 - 1/100 000. |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/250 | Notes - |
Species Rat | Dilution info 1/250 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info Use at an assay dependent concentration. | Notes - |
Select an associated product type
GFAP, a class-III intermediate filament, is a cell-specific marker that, during the development of the central nervous system, distinguishes astrocytes from other glial cells.
Glial fibrillary acidic protein, GFAP, GFAP
Anti-GFAP antibody [EPR1034Y] ab68428 is a rabbit monoclonal antibody that is used in GFAP western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests- Validated for multiplex IHC on the Leica BOND® MAX using Opal reagents; and for VCAM1 IHC using Leica BOND™ RX- Antibody clone EPR1034Y has been tried and trusted by researchers since 2008 and is cited in >180 publications- One antibody for all your GFAP staining, use in GFAP western blotting, IHC, immunofluorescence and flow cytometryNew 20 ul size available
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.21% BSA
Liquid
Monoclonal
EPR1034Y
Affinity purification Protein A
Blue Ice
1-2 weeks
+4°C
-20°C
Upon delivery aliquot
Avoid freeze / thaw cycle
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
This product is a recombinant monoclonal antibody, which offers several advantages including:
For more information, read more on recombinant antibodies.
We have tested this species and application combination and it works. It is covered by our product promise.
We have not tested this specific species and application combination in-house, but expect it will work. It is covered by our product promise.
This species and application combination has not been tested, but we predict it will work based on strong homology. However, this combination is not covered by our product promise.
We do not recommend this combination. It is not covered by our product promise.
We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.
In the unlikely event of one of our products not working as expected, you are covered by our product promise.
Full details and terms and conditions can be found here:
Terms & Conditions.
Fluorescence multiplex immunohistochemical analysis of human cerebellum tissue (formalin-fixed paraffin-embedded section).
Merged staining of Neu-N (Anti-NeuN antibody [EPR12763] - Neuronal Marker ab177487; yellow; Opal™570), anti-beta III Tubulin (Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker ab52623; red; Opal™690) and anti-GFAP (ab68428; green; Opal™520).
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ kit.
The section was incubated in three rounds of staining with Anti-NeuN antibody [EPR12763] - Neuronal Marker ab177487 (1/1000 dilution), Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker ab52623 (1/200 dilution) and ab68428 (1/250 dilution); each using a separate fluorescent tyramide signal amplification system.
Sodium citrate antigen retrieval (pH 6.0) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.
DAPI (blue) was used as a nuclear counter stain.
ab68428 at 1/20 dilution immunoprecipitating GFAP in rat brain whole cell lysate observed at 50 KDa (lanes 1 and 2).
Lane 1 (input): Rat brain whole cell lysate 10ug
Lane 2 (+): ab68428 + Rat brain whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab68428 in Rat brain whole cell lysate
For western blotting, ab68428 was used followed by VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/10,000 dilution.
Blocking and Diluting buffer and concentration: 5% NFDM/TBST.
All lanes: Immunoprecipitation - Anti-GFAP antibody [EPR1034Y] (ab68428)
Predicted band size: 49 kDa
Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Mouse primary brain cells cells labelling GFAP with ab68428 at 1/500 dilution (0.1ug)/ Right compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) / Left isotype control. A Goat anti rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
IHC image of GFAP staining in a formalin fixed, paraffin embedded normal human hippocampus tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab68428 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neural mix culture cells labelling GFAP with ab68428 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. ab10062 anti-GFAP antibody [GF5] at 1/100 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2μg/ml) was used as a counterstain. The Nuclear counterstain was DAPI (Blue).
Blocking and Diluting buffer 5% NFDM/TBST
All lanes: Western blot - Anti-GFAP antibody [EPR1034Y] (ab68428) at 1/10000 dilution
Lane 1: Human cerebellum tissue lysate at 20 µg
Lane 2: Human brain tissue lysate at 10 µg
All lanes: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution
Predicted band size: 49 kDa
Observed band size: 48-50 kDa
Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebrum tissue labeling GFAP with ab68428 at 1/1000 dilution (2.011 µg/mL) followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (2 µg/mL) (Green). Positive staining on rat cerebrum is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (2 µg/mL).<\p>
Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).
IHC image of GFAP staining in a section of frozen normal human cerebral cortex performed on a Leica BONDTM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab68428, 1/250 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Blocking and Diluting buffer 5% NFDM/TBST
All lanes: Western blot - Anti-GFAP antibody [EPR1034Y] (ab68428) at 1/10000 dilution
Lane 1: Mouse cerebellum tissue lysate at 20 µg
Lane 2: Mouse brain tissue lysate at 20 µg
All lanes: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution
Predicted band size: 49 kDa
Observed band size: 50 kDa
Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum tissue labeling GFAP with ab68428 at 1/1000 dilution (2.011 µg/mL) followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (2 µg/mL) (Green). Positive staining on mouse cerebrum is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (2 µg/mL).<\p>
Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).
Blocking and Diluting buffer 5% NFDM/TBST
All lanes: Western blot - Anti-GFAP antibody [EPR1034Y] (ab68428) at 1/50000 dilution
Lane 1: Rat cerebellum tissue lysate at 20 µg
Lane 2: Rat brain tissue lysate at 20 µg
All lanes: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution
Predicted band size: 49 kDa
Observed band size: 50 kDa
Immunohistochemical analysis of paraffin-embedded mouse liver tissue sections labelling GFAP with purified ab68428 at a dilution of 1/500. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP) ab97051, Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0.
All lanes: Western blot - Anti-GFAP antibody [EPR1034Y] (ab68428) at 1/5000 dilution
Lane 1: Human brain lysate at 10 µg
Lane 2: Rat brain lysate at 10 µg
All lanes: HRP labelled Goat anti-Rabbit antibody at 1/2000 dilution
Predicted band size: 49 kDa
Observed band size: 50 kDa
Immunohistochemical analysis of paraffin-embedded mouse cerebral cortex tissue sections labelling GFAP with purified ab68428 at a dilution of 1/500. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP) ab97051, Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.
Immunohistochemical analysis of paraffin-embedded human colon tissue sections labelling GFAP with purified ab68428 at a dilution of 1/500. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP) ab97051, Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0.
Immunohistochemical analysis of paraffin-embedded human cerebral cortex tissue sections labelling GFAP with purified ab68428 at a dilution of 1/500. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP) ab97051, Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.
Immunohistochemical analysis of formalin-fixed paraffin-embedded mouse brain tissue section labelling GFAP with unpurified ab68428 at dilution of 1/250.
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Immunohistochemical analysis of formalin-fixed paraffin-embedded human brain (left) and human glioma (right) tissue sections labelling GFAP with unpurified ab68428 at dilution of 1/250.
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Fluorescence multiplex immunohistochemical analysis of the human cerebrum (Formalin/PFA-fixed paraffin-embedded sections).
Panel A: merged staining of anti-GFAP (magenta; Opal™690), anti-MAP2 (green; Opal™520) and anti-GPCR GPR17 (red; Opal™570) on human cerebrum.
Panel B: anti-MAP2 staining neurons.
Panel C: anti-GPCR GPR17 staining oligodendrocytes.
Panel D: anti-GFAP staining astrocytes.
The section was incubated in three rounds of staining: in the order of Anti-MAP2 antibody [EPR22641-106] ab254263, Anti-GPCR GPR17 antibody [EPR26422-118] ab314307, and ab68428 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded rat spinal cord tissue sections.
Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-GFAP (red; Opal™570) on rat spinal cord.
Panel B: anti-GPR17 staining oligodendrocytes in rat spinal cord.
Panel C: anti-P2RY12 staining microglia in rat spinal cord.
Panel D: anti-GFAP staining astrocytes in rat spinal cord.
The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at a 1/40000 dilution, and Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309 at a 1/1000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
Nuclear counterstaining with DAPI.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
This data was developed using Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309, the same antibody clone in a different buffer formulation.
Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded mouse cerebrum tissue sections.
Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-GFAP (red; Opal™570) on mouse cerebrum.
Panel B: anti-GPR17 staining oligodendrocytes in mouse cerebrum.
Panel C: anti-P2RY12 staining microglia in mouse cerebrum.
Panel D: anti-GFAP staining astrocytes in mouse cerebrum.
The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at a 1/140000 dilution, and Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309 at a 1/1000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Nuclear counterstaining with DAPI.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
This data was developed using Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309, the same antibody clone in a different buffer formulation.
Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded rat cerebrum tissue sections..
Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-GFAP (red; Opal™570) on rat cerebrum.
Panel B: anti-GPR17 staining oligodendrocytes in rat cerebrum.
Panel C: anti-P2RY12 staining microglia in rat cerebrum.
Panel D: anti-GFAP staining astrocytes in rat cerebrum.
The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at 1/140000 dilution , and Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309 at 1/1000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The secondary was Opal Polymer HRP Ms + Rb and nuclear counterstaining with DAPI.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded mouse cerebellum tissue sections.
Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-GFAP (red; Opal™570) on mouse cerebellum.
Panel B: anti-GPR17 staining oligodendrocytes in mouse cerebellum.
Panel C: anti-P2RY12 staining microglia in mouse cerebellum.
Panel D: anti-GFAP staining astrocytes in mouse cerebellum.
The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at a 1/40000 dilution, and Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309 at a 1/1000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
Nuclear counterstaining with DAPI.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
This data was developed using Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309, the same antibody clone in a different buffer formulation.
Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded mouse spinal cord tissue sections.
Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-GFAP (red; Opal™570) on mouse spinal cord.
Panel B: anti-GPR17 staining oligodendrocytes in mouse spinal cord.
Panel C: anti-P2RY12 staining microglia in mouse spinal cord.
Panel D: anti-GFAP staining astrocytes in mouse spinal cord.
The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at a 1/40000 dilution, and Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309 at a 1/1000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
Nuclear counterstaining with DAPI.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
This data was developed using Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309, the same antibody clone in a different buffer formulation.
Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded mouse hippocampus tissue sections.
Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-P2RY12 (green; Opal™520) and anti-GFAP (red; Opal™570) on mouse hippocampus.
Panel B: anti-GPR17 staining oligodendrocytes in mouse hippocampus.
Panel C: anti-P2RY12 staining microglia in mouse hippocampus.
Panel D: anti-GFAP staining astrocytes in mouse hippocampus.
The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-P2Y12 antibody [EPR26298-93] ab300140 at a 1/40000, and Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309 at a 1/1000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
Nuclear counterstaining with DAPI.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
This data was developed using Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309, the same antibody clone in a different buffer formulation.
Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human cerebrum tissue sections.
Panel A: merged staining of anti-GPR17 (magenta; Opal™690), anti-TMEM119 (green; Opal™520) and anti-GFAP (red; Opal™570) on human cerebrum.
Panel B: anti-GPR17 staining oligodendrocytes in human cerebrum.
Panel C: anti-TMEM119 staining microglia in human cerebrum.
Panel D: anti-GFAP staining astrocytes in human cerebrum.
The section was incubated in three rounds of staining: in the order of Anti-GPCR GPR17 antibody [EPR26423-34] ab316105 at a 1/500 dilution, Anti-TMEM119 antibody [EPR25865-89] ab306583 at a 1/2000 dilution, and Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309 at a 1/1000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Nuclear counterstaining with DAPI.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
This data was developed using Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309, the same antibody clone in a different buffer formulation.
Fluorescence multiplex immunohistochemical analysis of the human cerebellum (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-GFAP (ab68428, gray; Opal™690), anti-Myelin PLP (Anti-Myelin PLP antibody [EPR23504-106] ab254363, green; Opal™520) and anti-P2Y12 (Anti-P2Y12 antibody [EPR23511-72] ab254347 red; Opal™570) on human cerebellum. Panel B: anti-P2Y12 stained on microglial cells. Panel C: anti-Myelin PLP stained on myelin sheaths of oligodendrocytes. Panel D: anti-GFAP stained on astrocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab68428 (1/50 dilution), Anti-Myelin PLP antibody [EPR23504-106] ab254363 (1/2000 dilution), and Anti-P2Y12 antibody [EPR23511-72] ab254347 (1/1000 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.
Fluorescence multiplex immunohistochemical analysis of the human cerebrum (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-GFAP (ab68428, gray; Opal™690), anti-Myelin PLP (Anti-Myelin PLP antibody [EPR23504-106] ab254363, green; Opal™520) and anti-P2Y12 (Anti-P2Y12 antibody [EPR23511-72] ab254347, red; Opal™570) on human cerebrum. Panel B: anti-P2Y12 stained on microglial cells. Panel C: anti-Myelin PLP stained on myelin sheaths of oligodendrocytes. Panel D: anti-GFAP stained on astrocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab68428 (1/50 dilution), Anti-Myelin PLP antibody [EPR23504-106] ab254363 (1/2000 dilution), and Anti-P2Y12 antibody [EPR23511-72] ab254347 (1/1000 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.
Fluorescence multiplex immunohistochemical analysis of human cerebellum tissue (formalin/PFA-fixed paraffin-embedded section). Panel A: merged staining of anti-GFAP (ab68428, gray; Opal™690), anti-Myelin PLP (Anti-Myelin PLP antibody [EPR23504-106] ab254363, green; Opal™520) and anti-MAP2 (Anti-MAP2 antibody [EPR22641-106] ab254263, red; Opal™570) on human cerebellum tissue. Panel B: anti-MAP2 stained cell body and dendrites of neurons. Panel C: anti-Myelin PLP stained on myelin sheaths of oligodendrocytes. Panel D: anti-GFAP stained on astrocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND®RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab68428 (1/50 dilution), Anti-Myelin PLP antibody [EPR23504-106] ab254363 (1/2000 dilution), and Anti-MAP2 antibody [EPR22641-106] ab254263 (1/4000 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) was used for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.
Fluorescence multiplex immunohistochemical analysis of human cerebrum tissue (formalin/PFA-fixed paraffin-embedded section). Panel A: merged staining of anti-GFAP (ab68428, gray; Opal™690), anti-Myelin PLP (Anti-Myelin PLP antibody [EPR23504-106] ab254363, green; Opal™520) and anti-MAP2 (Anti-MAP2 antibody [EPR22641-106] ab254263, red; Opal™570) on human cerebrum tissue. Panel B: anti-MAP2 stained cell body and dendrites of neurons. Panel C: anti-Myelin PLP stained on myelin sheaths of oligodendrocytes. Panel D: anti-GFAP stained on astrocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND®RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab68428 (1/50 dilution), Anti-Myelin PLP antibody [EPR23504-106] ab254363 (1/2000 dilution), and Anti-MAP2 antibody [EPR22641-106] ab254263 (1/4000 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) was used for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.
Fluorescence multiplex immunohistochemical analysis of the Human cerebellum (Formalin/PFA-fixed paraffin-embedded sections).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Opal Polymer HRP Ms + Rb was used as a secondary antibody.DAPI (blue) was used as a nuclear counter stain.
Panel A: Merged staining of anti-GFAP (gray; Opal™690), anti-P2Y12 (green; Opal™520) and anti-MAP2 (red; Opal™570) on human cerebellum.
Panel B: Anti-MAP2 stained cell body and dendrites of neurons.
Panel C: Anti-P2Y12 stained on microglial cells.
Panel D: Anti-GFAP stained on astrocytes.
The section was incubated in three rounds of staining: in the order of ab68428, Anti-P2Y12 antibody [EPR23511-72] ab254347, and Anti-MAP2 antibody [EPR22641-106] ab254263 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BONDBOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
Fluorescence multiplex immunohistochemical analysis of the Human cerebrum (Formalin/PFA-fixed paraffin-embedded sections).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Opal Polymer HRP Ms + Rb was used as a secondary antibody.DAPI (blue) was used as a nuclear counter stain.
Panel A: Merged staining of anti-GFAP (gray; Opal™690), anti-P2Y12 (green; Opal™520) and anti-MAP2 (red; Opal™570) on human cerebrum.
Panel B: Anti-MAP2 stained cell body and dendrites of neurons.
Panel C: Anti-P2Y12 stained on microglial cells.
Panel D: Anti-GFAP stained on astrocytes.
The section was incubated in three rounds of staining: in the order of ab68428, Anti-P2Y12 antibody [EPR23511-72] ab254347, and Anti-MAP2 antibody [EPR22641-106] ab254263 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
Immunohistochemical analysis of formalin fixed paraffin embedded human cerebellum labelling GFAP with ab68428 at a dilution of 1/1000. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an ChromoMap DAB (RUO) IHC Detection Kit with anti rabbit HQ and anti HQ HRP. Heat mediated antigen retrieval was conducted for 24 min with DISCOVERY cell conditioning solution (CC1) 100°C, pH 8.5. ab68428 was incubated at 37°C for 16 min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.
Chromogenic multiplex immunohistochemical staining of FFPE normal human cerebellum tissue. Anti-NeuN antibody [EPR12763] - Neuronal Marker ab177487, anti-NeuN DAB chromogen. ab68428, anti-GFAP purple chromogen and Anti-Iba1 antibody [EPR16588] ab178846, anti- Iba1 teal chromogen plus haematoxylin counterstain.
Chromogenic immunostaining was performed on a Roche Ventana Discovery Ultra instrument. The section was deparaffinised and incubated with CC1 solution for 24min 100°C. Following this with 3 rounds of staining in the order of Anti-NeuN antibody [EPR12763] - Neuronal Marker ab177487 (1/600), Anti-Iba1 antibody [EPR16588] ab178846 (1/4000) ab68428 (1/1000). Between rounds of staining, antibody denaturation was conducted using Ultra CC2 solution for 8min at 100°C to avoid cross reactivity. Signal was developed with anti-rabbit HQ followed by anti-HQ HRP coupled with Chromomap DAB kit, Discovery purple or Discovery teal chromogens and haematoxylin II counterstain.
Immunofluorescence staining of GFAP using ab68428 in primary rat hippocampal mixed glia, (prepared from P2 rat hippocampal brain area, obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. SDPHP4m), DIV4. The cells were fixed with 100% MeOH (5 min), permeabilized with 0.1% Triton-X-100 (in PBS) for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab68428 at 0.1 μg/ml and Anti-GFAP antibody ab4674, Anti-GFAP antibody, at 1/1000 dilution. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Chicken IgY H&L (Alexa Fluor® 594) preadsorbed ab150176, Goat Anti-Chicken IgY H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown. The antibody ab68428 gave comparable results using 4% formaldehyde fixation (10 min).
This data was developed using Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse midbrain tissue staining THP2 with Anti-TPH2 antibody [EPR25100-29] ab288067 at a 1/2000 (0.315 ug/ml) dilution, P2Y12 with Anti-P2Y12 antibody [EPR26298-93] ab300140 at 1/40000 (0.013 ug/ml) dilution and GFAP with Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309 at 1/1000 ( 1.325 ug/ml) dilution.
Panel A: merged staining of anti-THP2 (green; Opal™520), anti-P2Y12 (grey; Opal™570) and anti-GFAP (magenta; Opal™690) on mouse midbrain.
Panel B: anti-THP2 staining the serotonergic neurons in mouse midbrain.
Panel C: anti-P2Y12 staining microglia in mouse midbrain.
Panel D: anti-GFAP staining astrocytes in mouse midbrain.
Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of ab2888067, Anti-P2Y12 antibody [EPR26298-93] ab300140 and Anti-GFAP antibody [EPR1034Y] - BSA and Azide free ab218309 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
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