Overview

  • Product name
  • Description
    Chicken polyclonal to GFAP
  • Host species
    Chicken
  • Tested applications
    Suitable for: ELISA, IHC-Fr, IHC-FoFr, IHC-P, Flow Cyt, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Chicken, Cow, Human, Rhesus monkey, Apteronotus leptorhynchus
    Predicted to work with: Mammals
  • Immunogen

    Recombinant full length protein corresponding to Human GFAP. Isotype 1 expressed in and purified from E. coli.

  • Positive control
    • ICC/IF: Rat astrocytes. WB: Rat and mouse brain lysate. Apteronotus leptorhynchus brain tissue lysate. IHC-P: Rat hippocampus tissue. Mouse brain tissue. IHC-Fr: Human brain tissue. IHC-PFA fixed frozen: Mouse hippocampal tissue. Rabbit eye tissue. Flow Cyt: Human brain cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab4674 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ELISA 1/5000.
IHC-Fr 1/1000 - 1/5000. Try this antibody at about between about 1:1,000 using fluorescent secondary antibodies or 1:5,000 using peroxidase or other enzyme linked methods.
IHC-FoFr 1/1000 - 1/5000. PubMed: 20098733Try this antibody at about between about 1:1,000 using fluorescent secondary antibodies or 1:5,000 using peroxidase or other enzyme linked methods.
IHC-P 1/200 - 1/1000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt 1/100.

ab37382 - Chicken polyclonal IgY, is suitable for use as an isotype control with this antibody.

We recommend using ab46969 goat anti-chicken IgY FITC secondary antibody.

WB 1/1000 - 1/5000. Predicted molecular weight: 50 kDa.

Expect to see a band at 55kDa and another at about 48kDa, apparently a breakdown product of the 55kDa band.

ICC/IF 1/1000 - 1/5000.

Try this antibody at about between about 1:1,000 using fluorescent secondary antibodies or 1:5,000 using peroxidase or other enzyme linked methods.

PubMed: 25418722

Target

  • Function
    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.
  • Tissue specificity
    Expressed in cells lacking fibronectin.
  • Involvement in disease
    Defects in GFAP are a cause of Alexander disease (ALEXD) [MIM:203450]. Alexander disease is a rare disorder of the central nervous system. It is a progressive leukoencephalopathy whose hallmark is the widespread accumulation of Rosenthal fibers which are cytoplasmic inclusions in astrocytes. The most common form affects infants and young children, and is characterized by progressive failure of central myelination, usually leading to death usually within the first decade. Infants with Alexander disease develop a leukoencephalopathy with macrocephaly, seizures, and psychomotor retardation. Patients with juvenile or adult forms typically experience ataxia, bulbar signs and spasticity, and a more slowly progressive course.
  • Sequence similarities
    Belongs to the intermediate filament family.
  • Post-translational
    modifications
    Phosphorylated by PKN1.
  • Cellular localization
    Cytoplasm. Associated with intermediate filaments.
  • Information by UniProt
  • Database links
  • Alternative names
    • wu:fb34h11 antibody
    • ALXDRD antibody
    • cb345 antibody
    • etID36982.3 antibody
    • FLJ42474 antibody
    • FLJ45472 antibody
    • GFAP antibody
    • GFAP_HUMAN antibody
    • gfapl antibody
    • Glial fibrillary acidic protein antibody
    • Intermediate filament protein antibody
    • wu:fk42c12 antibody
    • xx:af506734 antibody
    • zgc:110485 antibody
    see all

Images

  • GFAP antibody ab4674 was used with Tissue Clearing Kit ab243298 to penetrate, stain and clear a 500 μm section of rat brain.

    Learn more about tissue clearing kits, reagents, and protocols designed to make it easier to stain thick tissue sections and get more data from each valuable tissue section.

  • 10x and 63x z-stack images of the CA1 hippocampal region for each treatment are shown, with NeuN staining in green and GFAP staining in red.

    Two month-old wild-type mice were placed on either control (n = 10) or inhibitor diet (PLX3397, provided at 290 mg/kg chow; n = 14) for 21 d, causing the elimination of approximately 99% of microglia brain-wide.

    Fluorescent immunolabeling of the microglia followed a standard indirect technique (primary antibody followed by fluorescent secondary antibody). Brain tissue (sliced at 40 μm) was stained using the anti-ionized calcium-binding adapter molecule 1 (IBA1, polyclonal, rabbit) antibody (1:1000; Wako, Cat. #019–19741), mounted on slides, and coverslipped using Dapi Fluoromount-G (SouthernBiotech). Half brain images were obtained by stitching using a Zeiss AxioImager M2 upright microscope and Stereo Investigator software package from MicroBrightField. In addition, tissue was stained with anti-hexaribonucleotide binding protein-3 (NeuN, monoclonal, mouse) antibody (1:1000; Millipore; Cat. #MAB377) to label neurons and anti-glial fibrillary acidic protein (GFAP, polyclonal, chicken) antibody (1:500; Abcam; Cat. #ab4674) to label astrocytes, and 10x and 63x z-stack images obtained for each treatment using confocal microscopy.

     

  • Immunofluorescent analysis of a section of mouse hippocampus stained with ab4674 at a 1:5,000 dilution in green.

    Costained with a rabbit pAb to FOX3/NeuN dilution 1:5,000, in red. The blue is DAPI staining of nuclear DNA. Following transcardial perfusion with 4% paraformaldehyde, mouse brain was post fixed for 24 hours, cut to 45 μM, and free-floating sections were stained. The GFAP antibody stains a network of astroglial cells while the Fox3/NeuN antibody stains the nuclei and proximal perikarya of neurons.

  • IHC image of GFAP staining in a formalin-fixed, paraffin-embedded mouse normal brain tissue section.

    The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH 6). The section was incubated with ab4674 at 1/1000 dilution for 15 minutes at room temperature. A goat anti-chicken biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. The section was counterstained with haematoxylin and mounted with DPX.

  • Rat astrocytes stained with ab4674 (red). The anti-GFAP antibody was used at a dilution of 1:50 from affinity purified material using our standard fixation and staining procedure (in protocol section). Hoechst dye reveals nuclear DNA in blue.
  • All lanes : Anti-GFAP antibody (ab4674) at 1/5000 dilution

    Lane 1 : Rat whole brain lysate
    Lane 2 : Mouse whole brain lysate

    Predicted band size: 50 kDa

  • ab4674 staining GFAP in human brain cells by Flow Cytometry.

    Cells were grown in stem cell media, RHB-A, and collected using 0.05% trypsin + trypsin inhibitor. Cells were fixed in 80% Acetone for 5-10 minutes at -20°C. The sample was incubated with the primary antibody (1/100 in 10% FCS in PBS) for 20 minutes at 25°C. ab46969 a goat anti-chicken IgY FITC (1/100) was used as the secondary antibody
    Gating Strategy: Cells expressing GFAP

    See Abreview

  • Anti-GFAP antibody (ab4674) at 1/1000 dilution + Apteronotus leptorhynchus brain tissue lysate at 50 µg

    Secondary
    AlexaFluor®488-conjugated goat anti-chicken polyclonal IgG at 1/1000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 50 kDa
    Observed band size: 50 kDa


    Exposure time: 5 minutes

    See Abreview

  • ab4674 staining GFAP in mouse hippocampus tissue section by Immunohistochemistry (PFA perfusion fixed frozen sections).

    Tissue samples were fixed by perfusion with 4% PFA and blocked with 10% serum for 30 minutes at 25°C. The sample was incubated with primary antibody (1/500) for 16 hours at 25°C in 10% NGS in PBS + 0.1% TX100. An Alexa Fluor®488-conjugated Goat polyclonal to chicken IgG was used as secondary antibody at 1/400 dilution. Staining was intensified with 2-3 minutes of retrieval with trypsin at room temperature.

    See Abreview

  • ab4674 staining GFAP in rat primary astrocytes by ICC/IF (Immunocytochemistry/immunofluorescence).

    Cells were fixed with paraformaldehyde, permeabilized with 0.05% Triton X-100 and blocked with 5% serum for 20 minutes at 20°C. Samples were incubated with primary antibody (1/2000) for 24 hours at 4°C. ab6873, a goat anti-chicken IgY FITC (1/1000) was used as the secondary antibody.

    See Abreview

  • Frozen sectioned human brain stained for GFAP with ab4674 (1/500 dilution, 18 hours at 4°C) in immunohistochemical analysis.

    10 µm cryostat cut sections fixed with 4% PFA prior to staining for 10 minutes. Goat polyclonal anti-chicken-Alex-Fluor®647 was used as the secondary at a 1/250 dilution.

    Micrograph demonstrates astrocyte processes (blue) next to cerebral vessels (red).

    See Abreview

  • ab4674 staining GFAP in rabbit eye tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections).

    Tissue samples were fixed by perfusion with formaldehyde and blocked with BSA for 2 hours at 4°C. The sample was incubated with primary antibody (1/1000) at 4°C for 12 hours. ab150175, a goat anti-chicken IgY Alexa Fluor® 647 (1/1000), was used as the secondary antibody.

    See Abreview

  • IHC image of GFAP staining in a formalin fixed, paraffin embedded normal rat hippocampus tissue section.

    The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH 6). The section was incubated with ab4674 at 1/1000 dilution for 15 minutes at room temperature. A goat anti-chicken biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. The section was counterstained with haematoxylin and mounted with DPX.

  • ab4674 detecting recombinant GFAP by direct ELISA.

    Mouse recombinant GFAP protein was coated onto a microplate in carbonate coating buffer pH 9.6 for 1 hour at 37°C. Plate was blocked with 3% BSA for 1 hour at 37°C and incubated with the primary antibody (1/5000 in PBS + 1% Tween-20) for 1 hour at 37°C. An undiluted alkaline phosphatase Goat anti-chicken IgG polyclonal was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:
See all 177 Publications for this product

Customer reviews and Q&As

1-6 of 6 Q&A

Answer

That will probably be fine, if you are able to cut out the insert easily. You may want to practice with a spare insert, and also see if the insert/mounting medium/coverslip combination will allow you to capture a good image. You will want to use an aqueous mounting medium, and these can sometimes allow bubbles to form as they dry out, so capture your images soon after coverslipping if possible. I am assuming you are working with a microscope with objectives above the stage, rather than an inverted microscope.

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Answer

We don't have any data on the effects of that kind of temperature on this antibody. However the blood temperature of a healthy chicken is 41.8C, or about ˜107F, so 50C is not too far from the normal range. Also antibodies are unusually stable molecules which have a long half life. So I would be surprised if incubation at 50C would be much of a problem.

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Answer

The antibody is an IgY prep from egg yolk, and it is basically the same as an IgG prep from plasma or serum. So it contains both the specific GFAP antibody and other immunoglobulins present in the chicken. So of the 30mg/ml total protein concentration about 3 or so are likely to be GFAP specific. The buffer is PBS, so that is amine free and the prep contains no significant amount of non-immunoglobulin protein. So it should be possible to directly label the GFAP specific IgY, though a lot of non specific IgY will also be labeled. However this IgY is not likely to be a problem as it clearly does not interfere with the ability of the GFAP specific antibody to bind GFAP on blots, in tissue culture or in sections. So direct labeling should be possible.
The main issue I see is the possibilty of non-GFAP staining, and the unusually high immunogobulin concentration. Simply diluting the antibody solution to the optimal concentration for conjugation will address that, and the data and references on the datasheet vouch for the specificity of the antibody.

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Question
Answer

According to our records, ab4674 was proving difficult to use in IHC-Fr and we were in contact in order to help resolve the issue.

Looking at our correspondence, it appears that we are awaiting more details in order to help us better understand the difficulties experienced. If the requested information has already been sent, it appears that it did not reach our Scientific Support team and we apologize for this inconvenience. In this case we would like to ask for the information again so that we can reach a resolution.

If the issue has already been settled, please let us know so that we can be assured that the problem has been solved to your satisfaction and update our records.

We wish you the best of luck with your research and look forward to a reply.

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Answer

BSA is added to the storage buffer when the antibody is a lower concentration. The additional protein provided by the addition of BSA stabilizes the antibody. This is unnecessary in case ofab4674 since this product shows a very high antibody concentration of 33mg/ml. BSA in the storage buffer is not connected to high background and unspecific binding during the experiment. Even when antibodies contain BSA we recommend to always include a blocking steop during the experiment. We do not have set expiration dates for our products. Most antibodies are stable and can last for anywhere from a few months to several years if stored properly, so we strongly recommend that you follow the storage instructions on the datasheet for the antibody you purchased. These conditions will vary among our antibodies, therefore, it is important to verify the storage conditions for each of our products when you receive them. We guarantee all of our products to work for at least twelve month from the date of purchase when stored correctly.

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Answer

This antibody does stain Bergmann glia in PFA fixed material, it should present no problems.

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