Rottlerin decreases the number of effector cells that mainly infiltrate the skin in IMQ-treated mice

Immunohistochemical detection of immune cell-related markers was performed on paraffin-embedded sections obtained from the back skin of IMQ-induced mice treated with vehicle or rottlerin.

Representatives IHC images of CD11b (B) on the skin of the vehicle or rottlerin-treated mice. Scale bar = 100μm.

Quantification analysis of IHC staining for CD11b(E) on the skin of the vehicle and rottlerin treated mice. Two independent researchers counted the number of positive staining cells were per high-power field (HPF). The data are representative of three experiments (n = 5 mice per group). ** P

All lanes : Anti-CD11b antibody [EPR1344] (ab133357) at 1/1000 dilution (purified)

Lane 1 : TF-1 cell lysate
Lane 2 : TPA treated THP-1 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 127 kDa
Observed band size: 170 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Formalin-fixed, 0.2% Triton-X permeabilized Mouse tissue sections (Eo771 breast cancer) was stained for CD11b using ab133357 (Green) at 1/1000 dilution in immunohistochemical analysis. The secondary antibody was a Alexa Fluor^® 488 Goat anti-Rabbit IgG (H+L) at 1/1000 dilution.

BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: 25°C

Ab133357 staining CD11b in paraffin embedded Mouse lung tissue sections by Immunohistochemistry. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at 1/4000 dilution (0.031 μg/ml). A ready to use Goat Anti-rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain. Positive staining on stromal cells of mouse lung.

Ab133357 staining CD11b in paraffin embedded Mouse colon tissue sections by Immunohistochemistry. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at 1/4000 dilution (0.031 μg/ml). A ready to use Goat Anti-rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain. Positive staining on stromal cells of mouse colon.

Ab133357 staining CD11b in paraffin embedded Rat cerebrum tissue sections by Immunohistochemistry. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at 1/4000 dilution (0.29 μg/ml). A ready to use Goat Anti-rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain. Positive staining on gliocytes of rat cerebrum [PMID: 20483006].

Formaldehyde-fixed, paraffin-embedded rat bone marrow tissue stained for CD11b using ab133357 at 1/5000 in immunohistochemical analysis.

Heat mediated antigen retrieval with EDTA buffer pH 9 was performed before commencing with staining protocol. 1% casein was used as blocking agent.

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IHC image of CD11b staining in a formalin fixed, paraffin embedded human normal spleen 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 ab133357 at 1/4000 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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Anti-CD11b antibody [EPR1344] (ab133357) at 1/1000 dilution + Rat spleen tissue lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 127 kDa
Observed band size: 170 kDa why is the actual band size different from the predicted?


Exposure time: 8 seconds

Blocking and diluting buffer: 5% NFDM/TBST

All lanes : purified at 1/10000 dilution

Lane 1 : RAW264.7 cell lysate
Lane 2 : Mouse spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 127 kDa
Observed band size: 170 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded human spleen with purified ab133357 at a 1/4000 dilution. The secondary antibody used is a HRP goat anti-rabbit (ab97051). The sample is counterstained with hematoxylin.

Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control, and is shown in the inset.