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, 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.

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, 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.

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

Lane 1 : Mouse testis
Lane 2 : Mouse liver
Lane 3 : Rat testis
Lane 4 : Rat liver

Predicted band size: 17 kDa

Flow cytometry analysis of 2% paraformaldehyde fixed U937 (human histiocytic lymphoma cell line) cells labeling Iba1 with 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).

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.

All lanes : 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: 17 kDa


Exposure time: 1 minute

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 ab97040 (HRP-labelled goat anti-mouse IgG) at  1:50,000 dilution for 1 hour at room temperature and visualised using ECL development solution ab133406.

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.

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

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

Lane 1 : THP-1 (human monocytic leukemia cell line) whole cell lysate
Lane 2 : U937 (human histiocytic lymphoma cell line) whole cell lysate
Lane 3 : Human spleen whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 17 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM /TBST

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.

Anti-Iba1 antibody [EPR16588] (ab178846) at 1/2000 dilution + HL-60 (human promyelocytic leukemia cell line) whole cell lysate at 10 µg

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

Predicted band size: 17 kDa
Observed band size: 10, 15 kDa why is the actual band size different from the predicted?

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.