Anti-Iba1 antibody [EPR16588]

19 product images

• 

  This image was courtesy of an annonymous customer review

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] (ab178846)

  Iba1 immunohistochemistry staining of monkey brain using rabbit anti-Iba1 antibody
  
  Formaldehyde-fixed paraffin-embedded cynomolgus monkey brain tissue stained for Iba1 using ab178846 at 1/6000 dilution in immunohistochemical analysis.

  Antigen Retrieval: Heat mediated - Buffer/Enzyme Used: pH 9.0 EDTA

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] (ab178846)

  Iba1 immunohistochemistry staining of rat brain using rabbit anti-Iba1 antibody
  
  IHC image of Iba1 staining in rat normal brain formalin fixed paraffin embedded 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 ab178846 at 1/2000 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.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] (ab178846)

  Iba1 immunohistochemistry staining of human hippocampus using rabbit anti-Iba1 antibody
  
  IHC image of Iba1 staining in human normal hippocampus formalin fixed paraffin embedded 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 ab178846 at 1/2000 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.

• 

  Western blot - Anti-Iba1 antibody [EPR16588] (ab178846)

  All lanes: Western blot - Anti-Iba1 antibody [EPR16588] (ab178846)

  Lane 1: Mouse testis

  Lane 2: Mouse liver

  Lane 3: Rat testis

  Lane 4: Rat liver

  Predicted band size: 16 kDa

• 

  Flow Cytometry (Intracellular) - Anti-Iba1 antibody [EPR16588] (ab178846)

  Iba1 flow cytometry staining of U937 cells using rabbit anti-Iba1 antibody
  
  Intracellular Flow Cytometry analysis of 2% paraformaldehyde fixed U937 (human histiocytic lymphoma cell line) cells labeling Iba1with ab178846 at 1/160 dilution (red line). Secondary antibody used is a goat anti rabbit IgG (FITC) at 1/150 dilution. The isotype control is rabbit monoclonal IgG (black line). The unlabeled control is cells without incubation with primary and secondary antibodies (blue line).

• 

  Western blot - Anti-Iba1 antibody [EPR16588] (ab178846)

  Different batches of ab178846 were tested on THP-1 (Human monocytic leukemia monocyte) lysate at 0.02 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 15 kDa.

  All lanes: Western blot - Anti-Iba1 antibody [EPR16588] (ab178846)

  Predicted band size: 16 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] (ab178846)

  IHC image of Iba1 staining in mouse normal brain formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. 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 ab178846, 1/2000 dilution, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC 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.

• 

  Western blot - Anti-Iba1 antibody [EPR16588] (ab178846)

  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 ab178846 (anti-Iba1 antibody; 1/500 dilution) for 18 hours at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (HRP) preadsorbed ab97040 (HRP-labelled goat anti-mouse IgG) at 1:50,000 dilution for 1 hour at room temperature and visualised using ECL development solution ECL Substrate Kit (High Sensitivity) ab133406.

  All lanes: Western blot - Anti-Iba1 antibody [EPR16588] (ab178846) at 1/500 dilution

  Lane 1: Human Iba1 recombinant protein at 0.1 µg

  Lane 2: HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

  Lane 3: A431 (human epidermoid carcinoma cell line) whole cell lysate at 20 µg

  Lane 4: NIH/3T3 (mouse embyro fibroblast cell line) whole cell lysate at 30 µg

  Lane 5: Human spleen tissue lysate at 20 µg

  Lane 6: Mouse spleen tissue lysate at 30 µg

  Lane 7: Rat spleen tissue lysate at 30 µg

  Lane 8: U937 (human histiocytic lymphoma cell line) whole cell lysate at 30 µg

  Lane 9: MOLT-4 (human lymphoblastic leukemia cell line) whole cell lysate at 20 µg

  Lane 10: THP-1 (human monocytic leukemia cell line) whole cell lysate at 30 µg

  Lane 11: THP-1 whole cell lysate, PMA treated at 30 µg

  Lane 12: RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 30 µg

  Lane 13: C6 (rat glial tumor cell line) 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: 16 kDa

  Exposure time: 1min

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16588] (ab178846)

  Iba1 immunofluorescence staining of microglia cells using rabbit anti-Iba1 antibody
  
  0.1% Triton-X 100 permeabilized paraformaldehyde-fixed Mouse cell Microglia cells labeling Iba1 (green) using ab178846 at 1/500 dilution in ICC/IF analysis.

• 

  Western blot - Anti-Iba1 antibody [EPR16588] (ab178846)

  Blocking buffer and concentration: 5% NFDM/TBST

  Diluting buffer and concentration: 5% NFDM /TBST

  All lanes: Western blot - Anti-Iba1 antibody [EPR16588] (ab178846) at 1/10000 dilution

  Lane 1: THP-1 (human monocytic leukemia cell line) whole cell lysate at 10 µg

  Lane 2: U937 (human histiocytic lymphoma cell line) whole cell lysate at 10 µg

  Lane 3: Human spleen whole cell lysate at 10 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

  Developed using the ECL technique.

  Predicted band size: 16 kDa

  Observed band size: 15 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] (ab178846)

  Immunohistochemical analysis of paraffin-embedded Human cerebral cortex tissue labeling Iba1 with ab178846 at a 1/2000 dilution showing cytoplasm and nuclear staining on Glial cells. Counter stained with hematoxylin. Prediluted HRP Polymer for Rabbit/Mouse IgG was used as the secondary aantibody. Negative control also shown.
  

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Western blot - Anti-Iba1 antibody [EPR16588] (ab178846)

  Blocking buffer and concentration: 5% NFDM/TBST.

  Diluting buffer and concentration:

  5% NFDM /TBST.

  Based on sequence analysis, ab178846 recognizes 2 isoforms with the predicted MWs of 17KDa and 11KDa, respectively.

  All lanes: Western blot - Anti-Iba1 antibody [EPR16588] (ab178846) at 1/2000 dilution

  All lanes: HL-60 (human promyelocytic leukemia cell line) whole cell lysate at 10 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

  Predicted band size: 16 kDa

  Observed band size: 10 kDa, 15 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16588] (ab178846)

  Iba1 immunofluorescence staining of primary rat hippocampal mixed glia cells using rabbit anti-Iba1 antibody
  
  Immunofluorescence staining of Iba-1 using ab178846 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 4% formaldehyde (10 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 ab178846 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 ab178846 gave comparable results using MeOH fixation (100% 5 min).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] (ab178846)

  Iba1 immunohistochemistry staining of human cerebellum using rabbit anti-Iba1 antibody
  
  Immunohistochemical analysis of formalin fixed paraffin embedded human cerebellum labelling Iba1 with ab178846 at a dilution of 1/4000. 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. ab178846 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.

• 

  Western blot - Anti-Iba1 antibody [EPR16588] (ab178846)

  Blocking/Dilution buffer: 5% NFDM/TBST.
  
  IBA1 is a relatively minor protein of brain and is much more abundant in spleen (PMID: 8912632, PMID: 29232670). We suggest loading higher amount of brain lysate or using lower dilution of antibody for detecting signal in brain related lysates.

  All lanes: Western blot - Anti-Iba1 antibody [EPR16588] (ab178846) at 1/1000 dilution

  Lane 1: Mouse spleen tissue lysate at 20 µg

  Lane 2: Mouse brain tissue lysate at 20 µg

  Lane 3: Mouse hippocampus tissue at 20 µg

  Lane 4: Rat spleen tissue lysate at 20 µg

  Lane 5: Rat brain tissue lysate at 20 µg

  Lane 6: Rat hippocampus tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - PE Anti-ENPP3/B10 antibody [NP4D6] (PE Anti-ENPP3/B10 antibody [NP4D6] ab90751) at 1/20000 dilution

  Predicted band size: 16 kDa

  Observed band size: 17 kDa

  Exposure time: 40s

• 

  Multiplex immunohistochemistry - Anti-Iba1 antibody [EPR16588] (ab178846)

  Chromogenic multiplex immunohistochemical staining of FFPE normal human cerebellum tissue. Anti-NeuN antibody [EPR12763] - Neuronal Marker ab177487, anti-NeuN DAB chromogen. Anti-GFAP antibody [EPR1034Y] ab68428, anti-GFAP purple chromogen and 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), ab178846 (1/4000) Anti-GFAP antibody [EPR1034Y] 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.

• 

  Flow Cytometry (Intracellular) - Anti-Iba1 antibody [EPR16588] (ab178846)

  Iba1 flow cytometry staining of Raw264.7 and NIH3T3 cells using rabbit anti-Iba1 antibody
  
  Flow cytometry overlay histogram showing left Raw264.7 positive cells and right negative NIH3T3 stained with ab178846 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab178846) (1x 106 in 100μl at 0.2μg/ml (1/9850 dilution)) for 30min at 22°C.The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°CIsotype control antibody (black line) was Recombinant Rabbit IgG monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• 

  Western blot - Anti-Iba1 antibody [EPR16588] (ab178846)

  Blocking buffer and concentration: 5% NFDM/TBST.
  
  Diluting buffer and concentration: 5% NFDM/TBST.
  
  IBA1 is a relatively minor protein of brain and is much more abundant in spleen (PMID: 8912632, PMID: 29232670). We suggest loading higher amount of brain lysate or using lower dilution of antibody for detecting signal in brain related lysates.
  
  Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as loading control.

  All lanes: Western blot - Anti-Iba1 antibody [EPR16588] (ab178846) at 1/1000 dilution

  Lane 1: Mouse spleen tissue lysate at 20 µg

  Lane 2: Mouse brain tissue lysate at 20 µg

  Lane 3: Mouse hippocampus tissue lysate at 20 µg

  Lane 4: Rat spleen tissue lysate at 20 µg

  Lane 5: Rat brain tissue lysate at 20 µg

  Lane 6: Rat hippocampus tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 16 kDa

  Observed band size: 17 kDa

  Exposure time: 40s

• 

  IHC-tissue clearing - Anti-Iba1 antibody [EPR16588] (ab178846)

  Iba1 immunohistochemistry staining of whole mouse brain using rabbit anti-Iba1 antibody
  
  Anti-Iba1 ab178846 was used with Tissue clearing kit – CUBIC (Tissue Clearing Kit – CUBIC ab316246) and 3D Tissue Staining Kit – CUBIC (3D Tissue Staining Kit – CUBIC ab316248) to penetrate stain and clear a whole mouse brain.

  Learn more about tissue clearing kits reagents and protocols designed to make it easier to stain whole brains and get more data from each valuable tissue sample.

  For a whole mouse brain we recommend starting with 3.5 ug of ab178846 and using a Fab fragment secondary antibody with 2.34 µg to create an antibody complex before 3D staining (see protocol for details). Additive A was used during the staining process.

  The sample was imaged using a light-sheet microscope.