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 416 Publications for this product

    Customer reviews and Q&As

    31-40 of 107 Abreviews or Q&A

    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Rat Tissue sections (Spinal cord L4,L5 avulsion injury 16 micron thick)
    Permeabilization
    No
    Specification
    Spinal cord L4,L5 avulsion injury 16 micron thick
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
    Fixative
    Formaldehyde

    Mr. Tamas Bellak

    Verified customer

    Submitted Dec 07 2015

    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.

    Read More
    This product is known to not work in this application or species.
    Application
    Immunohistochemistry (PFA perfusion fixed frozen sections)
    Sample
    Mouse Tissue sections (brain)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: sodium citrate
    Permeabilization
    No
    Specification
    brain
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Oct 05 2015

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Pig Tissue sections (Retina)
    Antigen retrieval step
    Heat mediated
    Permeabilization
    Yes - Tween
    Specification
    Retina
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
    Fixative
    Paraformaldehyde

    Dra. Marta Garcia Ramirez

    Verified customer

    Submitted Jul 08 2015

    This product is known to not work in this application or species.
    Application
    Immunohistochemistry (PFA perfusion fixed frozen sections)
    Sample
    Rat Tissue sections (brain)
    Antigen retrieval step
    None
    Permeabilization
    No
    Specification
    brain
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
    Fixative
    Paraformaldehyde

    Dr. Keerthi Thirtamara Rajamani

    Verified customer

    Submitted Jun 15 2015

    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (Mouse striatal section)
    Permeabilization
    Yes - Triton X-100 .3%
    Specification
    Mouse striatal section
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted May 01 2015

    This product is known to not work in this application or species.
    Application
    Immunohistochemistry (PFA perfusion fixed frozen sections)
    Blocking step
    Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Antigen retrieval step
    None
    Sample
    Mouse Tissue sections (brain)
    Specification
    brain
    Permeabilization
    Yes - triton 0.75%
    Fixative
    Paraformaldehyde

    Mrs. Francoise Geffroy

    Verified customer

    Submitted Mar 25 2015

    This product is known to not work in this application or species.
    Application
    Immunohistochemistry (PFA perfusion fixed frozen sections)
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
    Antigen retrieval step
    None
    Sample
    Mouse Tissue sections (Mouse brain (cortex shown in the figure but staini)
    Specification
    Mouse brain (cortex shown in the figure but staini
    Permeabilization
    Yes - 0.2% Triton X-100 during blocking, antibodies incubations
    Fixative
    Paraformaldehyde

    Dr. Jorge Valero

    Verified customer

    Submitted Oct 27 2014

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Blocking step
    BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Citric acid
    Sample
    Pig Tissue sections (Small intestine)
    Specification
    Small intestine
    Permeabilization
    No
    Fixative
    Formaldehyde

    Mr. Carl Hobbs

    Verified customer

    Submitted Sep 05 2014

    This product is known to not work in this application or species.
    Application
    Immunohistochemistry (PFA perfusion fixed frozen sections)
    Blocking step
    Donkey serum +bsa as blocking agent for 2 hour(s) and 0 minute(s) · Temperature: 22°C
    Antigen retrieval step
    None
    Sample
    Rat Tissue sections (brain)
    Specification
    brain
    Permeabilization
    Yes - triton 0.2%
    Fixative
    Paraformaldehyde

    Mrs. Francoise Geffroy

    Verified customer

    Submitted Aug 21 2014

    31-40 of 107 Abreviews or Q&A

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