Anti-Iba1 antibody (ab5076)

Goat polyclonal Iba1 antibody. Validated in WB, IHC, ICC, EM, ICC/IF and tested in Rat, Rabbit, Guinea pig, Cow, Dog, Human, Pig, Common marmoset. Cited in 316 publication(s). Independently reviewed.

Overview

  • Product name

  • Description

    Goat polyclonal to Iba1
  • Host species

    Goat
  • Specificity

    Iba1 antibody (ab5076) is expected to recognise the isoforms represented by NP_001614 and NP_116573 but not NP_004838.  Please note: Although we have some very good Abreviews on mouse and pig, some customers were receiving inconsistent results on mouse samples. We have therefore removed mouse and pig and can no longer guarantee them. In addition, we recommend blocking in milk. Blocking with BSA gives high background.

    IHC-Fr: Some customers have used ab5076 in IHC-Fr however we have had consistent complaints in this application over the last year and can no longer guarantee performance in this application. If looking for a monoclonal anti-Iba1 alternative we can recommend our RabMAb, Iba1 antibody [EPR16588] (ab178846) which has the same immunogen as ab5076.

  • Tested applications

    Suitable for: Electron Microscopy, IHC-P, WB, IHC-FrFl, ICC, ICC/IFmore details
    Unsuitable for: IHC-FoFr
  • Species reactivity

    Reacts with: Rat, Rabbit, Guinea pig, Cow, Dog, Human, Common marmoset
    Predicted to work with: Macaque monkey
  • Immunogen

    Synthetic peptide corresponding to Human Iba1 aa 135-147 (C terminal). Accession Number(s): NP_116573.1; NP_001614.3
    Sequence:

    C-TGPPAKKAISELP


    (Peptide available as ab23067)

  • Positive control

    • Recombinant Human Iba1 protein (ab117478) can be used as a positive control in WB.
  • General notes

    The Iba1 antibody (ab5076) is commonly used as a marker of microglia activation in staining and immunohistochemistry, given that ionized calcium binding adaptor molecule 1 (Iba1) is a microglia/macrophage-specific calcium-binding protein with actin-bundling activity that participates in membrane ruffling and phagocytosis in activated microglia.

Properties

Applications

Our Abpromise guarantee covers the use of ab5076 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
Electron Microscopy Use at an assay dependent concentration. PubMed: 26358247
IHC-P Use a concentration of 2 - 4 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use a concentration of 0.3 - 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 17 kDa).Can be blocked with Human Iba1 peptide (ab23067).

We received several excellent Abreviews on this antibody working with mouse, however, mouse is not a batch tested species and we cannot guarantee that this antibody will work on mouse. Some of our customers have observed high background in mouse samples. We recommend blocking in milk. Blocking with BSA gives high background.

IHC-FrFl Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
  • Application notes
    Is unsuitable for IHC-FoFr.
  • Target

    • Function

      Actin-binding protein that enhances membrane ruffling and RAC activation. Enhances the actin-bundling activity of LCP1. Binds calcium. Plays a role in RAC signaling and in phagocytosis. May play a role in macrophage activation and function. Promotes the proliferation of vascular smooth muscle cells and of T-lymphocytes. Enhances lymphocyte migration. Plays a role in vascular inflammation.
    • Tissue specificity

      Detected in T-lymphocytes and peripheral blood mononuclear cells.
    • Sequence similarities

      Contains 2 EF-hand domains.
    • Post-translational
      modifications

      Phosphorylated on serine residues.
    • Cellular localization

      Cytoplasm > cytoskeleton. Cell projection > ruffle membrane. Associated with the actin cytoskeleton at membrane ruffles and at sites of phagocytosis.
    • Information by UniProt
    • Database links

    • Alternative names

      • AIF 1 antibody
      • AIF-1 antibody
      • Aif1 antibody
      • AIF1 protein antibody
      • AIF1_HUMAN antibody
      • Allograft inflammatory factor 1 antibody
      • Allograft inflammatory factor 1 splice variant G antibody
      • allograft inflammatory factor-1 splice variant Hara-1 antibody
      • balloon angioplasty responsive transcription antibody
      • BART 1 antibody
      • G1 antibody
      • G1 putative splice variant of allograft inflamatory factor 1 antibody
      • IBA 1 antibody
      • IBA1 antibody
      • interferon gamma responsive transcript antibody
      • Interferon responsive transcript 1 antibody
      • interferon responsive transcript factor 1 antibody
      • Ionized calcium binding adapter molecule 1 antibody
      • Ionized calcium-binding adapter molecule 1 antibody
      • ionized calcium-binding adapter molecule antibody
      • IRT 1 antibody
      • IRT1 antibody
      • Microglia response factor antibody
      • MRF1 antibody
      • Protein g1 antibody
      see all

    Images

    • IHC-P image of Iba1 staining on cow kidney sections using ab5076 (1:2000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 mins at 21°C. ab5076 was diluted 1:2000 and incubted with the sections for 2 hours at 21°C. The secondary antibody used was Rabbit polyclonal to anti sheep/goat IgG conjugated to biotin (1:200)

      See Abreview

    • ab5076 staining Iba1 in the Macrophage cell line Mono-Mac 6 by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and blocked with 10% serum for 20 minutes at 24°C. Samples were incubated with primary antibody (1/500 in PBS) for 16 hours at 4°C. An Alexa Fluor® 555-conjugated Rabbit anti-goat IgG polyclonal (1/1000) was used as the secondary antibody.

      See Abreview

    • Lane 1 : Anti-Iba1 antibody (ab5076) at 1 µg
      Lane 2 : Anti-Iba1 antibody (ab5076) at 0.3 µg

      Lane 1 : Human Frontal Cortex lysate
      Lane 2 : Rat Brain lysate

      Lysates/proteins at 35 µg per lane.

      Predicted band size: 17 kDa

    • IHC-P image of Iba1 staining on cat kidney sections using ab5076 (1:1000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 mins at 21°C. ab5076 was diluted 1:1000 and incubted with the sections for 2 hours at 21°C. The secondary antibody used was Rabbit polyclonal to anti sheep/goat IgG conjugated to biotin (1:200)

      See Abreview

    • IHC-P image of Iba1 staining on marmoset bladder sections using ab5076 (1:1000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 mins at 21°C. ab5076 was diluted 1:1000 and incubted with the sections for 2 hours at 21°C. The secondary antibody used was Rabbit polyclonal to anti goat IgG conjugated to biotin (1:200)

      See Abreview

    • All lanes : Anti-Iba1 antibody (ab5076) at 1 µg/ml

      Lane 1 : Human Iba1 full length recombinant protein at 0.1 µg
      Lane 2 : HEK293 whole cell lysate at 30 µg
      Lane 3 : A431 whole cell lysate at 30 µg
      Lane 4 : NIH3T3 whole cell lysate at 30 µg
      Lane 5 : Human spleen tissue lysate at 30 µg
      Lanes 6 & 15 : Mouse spleen tissue lysate. (Due to inconsistent results we can no longer recommend this antibody for use in mouse) at 30 µg
      Lane 7 : Rat spleen tissue lysate at 30 µg
      Lane 8 : U937 whole cell lysate at 30 µg
      Lane 9 : HL60 whole cell lysate at 30 µg
      Lanes 10 & 16 : THP1 whole cell lysate at 30 µg
      Lane 11 : THP1 whole cell lysate, PMA treated at 30 µg
      Lane 12 : Raw 264.7 whole cell lysate at 30 µg
      Lane 13 : C6 whole cell lysate at 30 µg
      Lane 14 : NR8383 whole cell lysate at 30 µg

      Developed using the ECL technique.

      Performed under reducing conditions.

      Predicted band size: 17 kDa



      Lanes 1-14: Blocked in 3% milk for 1 hour (RT). Lanes 15-16: Blocked in 5% BSA for 1 hour (RT).

      Lane 1: exposure 1 minute. Lanes 2-16: exposure 4 minutes. Abcam recommends blocking in milk for cleaner blots with reduced background, in comparison to BSA. 

      This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab5076 (anti-Iba1 antibody; 1 ug/mL) for 18 hours at 4°C. 

    • Immunohistochemical analysis of formalin-fixed, paraffin-embedded human lateral prefrontal cortex tissue labeling iba1 labeled microglia (labeled with black arrows) within the cortex using ab5076 at 1/1000 dilution. (Cropped image).

      Sections were rinsed in 0.01 m PBS, pH 7.4, followed by 10% serum matching the species of the secondary antibody, 5% bovine serum albumin, and 0.1% Triton X-100 in 0.01 m PBS blocking solution for 1 h and incubated for 2 days at 4 °C in primary antibody. The sections were rinsed in PBS and incubated overnight at 4 °C with a horse anti-Goat IgG (Biotinylated) secondary antibody.

    • ab5076 staining Iba1 in rat glioblastoma cell line C6 by Immunocytochemistry/ Immunofluorescence.Cells were fixed in paraformaldehyde, permeabilized using 0,1% Triton X 100 in PBS, blocked with 0.5% BSA for 30 minutes at room temperature and then incubated with ab5076 at a 1/50 dilution for 16 hours at 4°C. The secondary used was a Cy3 conjugated rabbit anti-goat polyclonal used at a 1/120 dilution. Nuclei are counterstained with DAPI.

      See Abreview

    • ab5076 staining Iba1 in guinea pig brain tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent formaldehyde fixation before heat mediated antigen retrieval in sodium citrate and then blocking with 3% serum was performed for 30 minutes at RT. The primary antibody was used at dilution at 1/200 and incubated with sample at 2% blocking serum for 18 hours at 4°C. A Biotin conjugated horse polyclonal to goat IgG was used undiluted as secondary antibody.

      See Abreview

    • AIF1 expression in an inflammatory foci in a skin biopsy. Paraffin embedded sections were deparaffinized and boiled in 10mM citrate buffer for 30 min in a microwave to expose the AIF1 antigen, and then blocked with 5% donkey serum for 20 min. Slides were incubated for 40 min with goat-anti-AIF1 antibody (1:100) and rinsed in three changes of PBS for 1 min each.  A secondary antibody (donkey-anti-goat-FITC conjugated IgG; 1:50) was then applied to the slide for 40 min. Sections were washed again to remove the unbound antibody. The slides were counterstained with DaPI and viewed with a Nikon epi-fluorescent microscope. AIF1 positive cells appear green and the nuclei are stained blue. Numerous cells expressing the AIF1 protein are located around a vessel. (400X). Picture from Rreview by Carol Artlett submitted 30 July 2004.
    • IHC-P image of Iba1 staining on rat brain sections using ab5076 (1:1000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 mins at 21°C. ab5076 was diluted 1:1000 and incubted with the sections for 2 hours at 21°C. The secondary antibody used was Rabbit polyclonal to anti sheep/goat IgG conjugated to biotin (1:200)

      See Abreview

    • Paraffin embedded sections of human lung were stained for Iba1 with ab5076 at 5 μg/ml in immunohistochemical analysis.

    • Free floating sections of rat striatum/microglia were stained for Iba1 with ab5076 at 1/2000 dilution in immunohistochemical analysis. Rabbit Anti-Goat IgG Biotin was used as the secondary antibody at 1/200 dilution.

    References

    This product has been referenced in:

    • Hussain T  et al. Wwox deletion leads to reduced GABA-ergic inhibitory interneuron numbers and activation of microglia and astrocytes in mouse hippocampus. Neurobiol Dis 121:163-176 (2019). Read more (PubMed: 30290271) »
    • Skowronska K  et al. Persistent Overexposure to N-Methyl-D-Aspartate (NMDA) Calcium-Dependently Downregulates Glutamine Synthetase, Aquaporin 4, and Kir4.1 Channel in Mouse Cortical Astrocytes. Neurotox Res 35:271-280 (2019). Read more (PubMed: 30220059) »
    See all 397 Publications for this product

    Customer reviews and Q&As

    1-10 of 25 Q&A

    Answer

    The molar concentration of the Tris is 20mM. The formulation of the buffer is Tris saline (20mM Tris pH7.3, 150mM NaCl), 0.02% sodium azide, with 0.5% bovine serum albumin.

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    Answer

    Thank you for contacting Abcam.

    I managed to find antibodies against rat GFAP, FOX3, Iba1, and CD68 all raised in different hosts (chicken, rabbit, goat, and mouse, respectively). They each are highly rated by other researchers and have numerous specific published references. The list for these 4 antibodies is given below:
    1. GFAP ab4674 (https://www.abcam.com/gfap-antibody-astrocyte-marker-ab4674.html)
    2. FOX3 ab104225 (https://www.abcam.com/fox3neun-antibody-ab104225.html)
    3. Iba1 ab5076 (https://www.abcam.com/iba1-antibody-ab5076.html)
    4. CD68 ab31630 (https://www.abcam.com/cd68-antibody-ed1-ab31630.html)

    I also compiled anti-chicken, rabbit, goat, and mouse secondaries with separate emission spectra. We don't have secondaries for these species that weren't raised in any of the 4 species conjugated to a fluorescent protein with a similar spectrum to DAPI. Thus, I think your best bet is to obtain a far-red filter (Cy5), that way you could still stain with DAPI as well. The donkey secondaries that I found with separate emission spectra are listed below:
    1. anti-chicken: FITC (https://www.abcam.com/chicken-igy-secondary-antibody-ab63507.html)
    2. anti-rabbit: DyLight 594 (Similar spectrum to Texas Red) (https://www.abcam.com/donkey-polyclonal-secondary-antibody-to-rabbit-igg-h-l-dylight-594-pre-adsorbed-ab96921.html)
    3. anti-goat: DyLight 650 (Similar spectrum to Cy5) (https://www.abcam.com/donkey-polyclonal-secondary-antibody-to-goat-igg-h-l-dylight-650-pre-adsorbed-ab96938.html)
    4. anti-mouse: DyLight 550 (similar spectrum to TRITC) (https://www.abcam.com/donkey-polyclonal-secondary-antibody-to-mouse-igg-h-l-dylight-550-ab96876.html)

    The other antibodies you chose (Nogo, heavy neurofilament, caspase 3) seem to be good choices as well as they are tested in formaldehyde fixed, frozen sections via IHC in rat. Below are the antibodies we discussed against those three proteins:
    1. anti-Nogo (https://www.abcam.com/nogo-antibody-ab32298.html)
    2. anti-heavy neurofilament (https://www.abcam.com/200-kd-neurofilament-heavy-antibody-ab134459.html)
    3. anti-caspase 3 (https://www.abcam.com/active-caspase-3-antibody-ab13847.html)

    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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    Answer

    Thank you for contacting us.

    Unfortunately, we do not have a negative control image for ICC at hand.

    However, we do have the peptide available, which can be used as as blocking peptide in this case to check if the astrocytes staining is specific.

    https://www.abcam.com/index.html?datasheet=23067 (or use the following: https://www.abcam.com/index.html?datasheet=23067).


    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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    Answer

    I am very pleased to hear you would like to test ab5076, ab8049, ab18258, ab58802, ab64581, ab82259 and send us your results. The testing discount doesn't apply to ab4674 since this has already been tested in Rhesus monkey and IHC so we would guarantee that experiment to work.

    The codes will give you a value off your next orders before the expiration date. To redeem this offer, please submit 6 Abreview with rhesus monkey data and include the code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
    For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

    The code will be active once the Abreview wit has been submitted and can be redeemed in one of the following ways:
    1) Call to place your order and mention the code to our customer service department;
    2) Include the code in your fax order;
    3) Place your order on the web and enter the promotional code.

    Any feedback that you can provide will be greatly appreciated. Remember if the product does not work you will be covered by our Abpromise guarantee and eligible for a full refund or replacement.

    If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

    The terms and conditions applicable to this offer can be found here: https://www.abcam.com/abtrial.

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    Answer

    Thank you for your enquiry.

    I would like to reassure you that our antibodies have all been tested successfully and would be covered by our guarantee in the applications listed on the datasheets. Therefore, any antibody tested in the species and application you are using should be suitable for your applications.

    Also, although we produce many in-house antibodies, we obtain other antibodies from a wide range of sources. Therefore, comparison experiments will not often have been done.

    If you wish to be more certain, I can suggest choosing the antibody that has been best characterized. By this I mean the antibody that has been tested in the most applications and has the most data available (on the datasheet).

    In regard to your concerns about ab15690 Anti-Iba1 antibody [1022-5], we do offer another Anti-Iba1 which has been raised in goat and has a number of referenced publications, Abreviews and support data. I would recommend this product as an alternative to ab15690.

    https://www.abcam.com/iba1-antibody-ab5076.html


    I look forward to hearing from you. Should you have any further questions please do not hesitate to ask.

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    Question
    Answer

    Thank you for contacting us.

    Please find the information below describingour testing discount program:

    For UNTESTED species and/or applications, we have established a testing discount program. Here is a brief description of how it works:

    The testing discount program is for customers who like to use an antibody/protein/kit on an untested species/application. You would purchase the antibody/protein/kit at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody/protein at the full price (100%) of the antibody/protein you have tested, or a full price discount for the amount paid for the kit. The terms and conditions applicable to this offer can be found here: https://www.abcam.com/collaborationdiscount.
    This programapplies to ELISA kits and other kits we offer.


    Also, I wanted to check homology betweenhuman and rat MMP1, but was not able to find the full length protein sequence for rat MMP1 in the database.
    However, if you like to try any of the antibodies that do not list "rat" as tested species (only as predicted), please let me know and I'd be happy to send you a testing discount code.

    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

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    Answer

    Thank you for contacting us.

    You can try any of ab97100, ab97110, ab97120, ab98519 etc.

    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

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    Question
    Answer

    For ab5076: DISCOUNT CODE: ***

    For ab110939: DISCOUNT CODE: ***

    Expiration date: December 1st, 2012

    I am very pleased to hear you would like to accept our offer and test ab5076 or ab110939on mouse samples. This code will give you: 1 freeprimary antibodybefore the expiration date. To redeem this offer, please submit an Abreview for mouseand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
    For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

    Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

    Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

    The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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    Answer

    Thank you for submitting an Abreview for ab5076. As you may have noticed, your review has now been published on our website.
    Since you obtained poor results using the antibody in an untested species, we would like to follow up on this to see if we can possibly improve the results you are seeing with this antibody.
    I'd like to offer some suggestions, but am not sure what the results were.
    Thus, I was wondering what the actual problem was with the antibody, e.g. no signal or high background.
    Also, if you like, you can add an image to show your findings and earn 100 extra Abpoints.
    I wish you good luck with your research.

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    Answer

    Thank you for taking the time to contact us. I am sorry to hearthe customer hashad difficulty obtaining satisfactory results from these threeantibodies.

    I would like to reassure you that in the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

    I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

    I would appreciate if you could also provide images which would help us to assess the results.

    Please note Ihave sent the questionnaire for IHC and ICC. If the customer has used another application, please let me know and I can send the correct form so we can obtain the relevant information required.

    Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.



    Order Details
    Antibody code:

    Lot number:

    Purchase order number
    or preferably Abcam order number:


    General Information
    Antibody storage conditions (temperature/reconstitution etc)


    Description of the problem (high background, low signal, non-specific satining etc.)

    Sample (Species/Tissue/Cell Type/Cell Line etc.)


    Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)


    Antigen retrieval (Enzymatic method, Heat mediated technique etc.)


    Permeabilization step


    Blocking conditions (Buffer/time period, Blocking agent etc.)


    Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


    Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


    Detection method


    Positive and negative controls used (please specify)


    Optimization attempts (problem solving)
    How many times have you tried the IHC?



    Have you run a "No Primary" control?
    Yes No

    Do you obtain the same results every time?
    Yes No


    What steps have you altered?


    Additional Notes


    We would appreciate if you are also able to provide and image which woudl help us to assess the results

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    1-10 of 25 Q&A

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