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 ab153696, 5µg/ml, 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.

Immunofluorescent analysis of HeLa cells (fixed in iced cold methanol; 5mins) labeling Iba1 with ab153696 at 1/500 dilution (left). Right image shows cells co-stained with Hoechst 33342.

All lanes : Anti-Iba1 antibody (ab153696) at 1/500 dilution

Lane 1 : K562 whole cell extracts
Lane 2 : THP-1 whole cell extracts
Lane 3 : HL-60 whole cell extracts

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : HRP-conjugated anti-rabbit IgG antibody at 1/10000 dilution

Developed using the ECL technique.

Predicted band size: 16 kDa

High expression in THP-1. Low expression in K562 and HL-60.

15% SDS-PAGE.

Running condition: 80V, 15min; 140V, 40 min.

Transfer condition: Semi-dry, 18 V, 60 min (Nitrocellulose membrane).

Blocking condition: 5% non-fat milk in TBST, RT, 60 min.

Primary antibody incubation: 4°C overnight.

Washing condition: 5 ml TBST, 4 x 5 min.

ECL exposure. 

Flow cytometric analysis of THP-1 (human monocytic leukemia cell line) cell line labeling Iba1 with ab153696 at 1/50 dilution (red) compared with an unlabelled sample (black).

The sample was fixed using 4% PFA in PBS at 4℃ for 15 minutes. Cells were resuspended twice in 0.1% Triton X-100 in PBS (wash buffer), then centrifuged at 4℃ for 5 mintues. The sample was incubated with the primary antibody (1/50 in PBS) for 60 minutes at 4°C. A. Alexa Fluor^® 488-conjugated anti-rabbit IgG (1/1000 in PBS) at 4^oC for 30 minutes was used as the secondary antibody.

Analysed using a BD Accuri™ C6 Cytometer.

IHC image of Iba1 staining in normal mouse 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 ab153696, 1µg/ml, 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.

Paraffin-embedded rat brain tissue stained for Iba1 using ab153696 at 1/500 dilution in immunohistochemical analysis. Antigen retrieval: Citrate buffer, pH 6.0, 15 min

 

ab221591 staining Iba1 in Mouse brain tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 1.5% goat serum (dilute goat serum by 1xPBS), at room temperature for 30min; antigen retrieval was by heat mediation in a citrate buffer, pH 6.0, 15min (Cuisinart Electric Pressure Cooker #EPC-1200, choose "high pressure"). Samples were incubated with primary antibody (1/500 in blocking buffer) for overnight at 4°C. An ABC HRP Kit (Rabbit IgG) was used as the secondary antibody (1:200), room temperature for 30min. Washed twicefor 5 minutes using PBS. DAB was used as a substrate.

Flow cytometic analysis of primary murine microglia cells labeling Iba1 with ab153696 at 1.0 µg per 4×10⁵ cells (blue), Rabbit IgG (green) , Unstained (red).

All lanes : Anti-Iba1 antibody (ab153696) at 1/500 dilution

Lane 1 : Human Iba1 full length recombinant protein at 0.1 µg
Lane 2 : HEK293 whole cell lysate at 20 µg
Lane 3 : A431 whole cell lysate at 20 µg
Lane 4 : NIH3T3 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 whole cell lysate at 30 µg
Lane 9 : MOLT4 whole cell lysate at 30 µg
Lane 10 : 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: 16 kDa


Exposure time: 1 minute

BLOCKED IN 3% MILK. For ab153696 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 ab153696 (anti-Iba1 antibody; 1/500 dilution) for 18 hours at 4°C. Antibody binding was detected using HRP-labelled anti-Rabbit IgG  for 1 hour at room temperature and visualised using ECL development solution ab133406.

Anti-Iba1 antibody (ab153696) at 1/1000 dilution + Rat liver extract at 50 µg

Secondary
HRP-conjugated anti-rabbit IgG antibody

Predicted band size: 16 kDa

15% SDS-PAGE

Anti-Iba1 antibody (ab153696) at 1/1000 dilution + Mouse liver whole cell lysate at 50 µg

Secondary
Rabbit IgG antibody (HRP) for 1 hour at room temperature at 1/10000 dilution

Predicted band size: 16 kDa

15% SDS PAGE

Running conditions: 80V for 15min then 140V for 40min

Blocking: 5% non-fat milk in TBST at room temperature for 60min.

Washing conditions: 5 ml TBST, 4 x 5min

Transfer conditions: Semi-dry, 18 V, 60min (NC membrane)

Exposure system: Trident plus Western HRP Substrate

All lanes : Anti-Iba1 antibody (ab153696) at 1/5000 dilution

Lane 1 : THP1 whole cell lysate
Lane 2 : HL60 whole cell lysate

Lysates/proteins at 30 µg per lane.

Predicted band size: 16 kDa



15% SDS PAGE