Recombinant Anti-CD11b antibody [EP1345Y] - C-terminal (ab52478)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical cancer tissue sections labeling CD11b with purified ab52478 at 1:1000 dilution (0.28 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0) ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Hematoxylin was used as a counterstain.

Negative control: PBS instead of the primary antibody (inset).

Unpurified ab52478 staining CD11b in the THP-1 (Human monocytic leukemia cell line) cell line by ICC/IF (Immunocytochemistry/immunofluorescence).

Cells were fixed with 100% methanol. Samples were incubated with primary antibody (1/250). ab150077 was used as the secondary antibody (1/1000). Nuclei were stained with DAPI.

Blocking and diluting buffer: 5% NFDM/TBST.

ab52478 (purified) at 1:30 dilution (2 µg) immunoprecipitating CD11b in TF-1 (Human bone marrow erythroleukemia cell line) whole cell lysate.
Lane 1: TF-1 whole cell lysate 10 µg (input). 
Lane 2: ab52478 + TF-1 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab52478 in TF-1 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Enhanced expression of monocytes/ macrophage markers in the obese adipose tissue.

The protein expression (intensity) of monocyte/ macrophage markers was detected by immunohistochemistry (IHC) in the adipose tissue samples from lean, overweight, and obese individuals, 10 each. As shown by representative IHC photomicrographs (100× magnification), expression of (A) CD11b was found to be markedly elevated in overweight and obese adipose tissue samples as compared with lean samples.

Paraffin-embedded sections (4 μm thick) of subcutaneous adipose tissue were deparaffinized in xylene and rehydrated through descending grades of ethanol (100%, 95%, and 75%) to water. Antigen retrieval was performed under pressure cooker boiling for 8 min and cooling for 15 min. After washing in PBS, endogenous peroxidase activity was blocked with 3% H₂O₂ for 30 min and non-specific antibody binding was clocked with 5% nonfat milk for 1hr and 1% bovine serum albumin (BSA) solution for 1hr. Slides were treated overnight with primary antibodies at room temperature. After washing with PBS (0.5% Tween), slides were incubated for 1hr with secondary antibody conjugated with HRP polymer chain and color was developed using 3,3ʹ-diaminobenzidine chromogen substrate. Specimens were washed in running tap water, lightly counterstained with hematoxylin, dehydrated through ascending grades of ethanol (75%, 95%, and 100%), cleared in xylene, and finally mounted in dibutyl phthalate xylene (DPX).

Duchenne muscular dystrophy (DMD) muscle was co-stained for Neu5Gc (green), ab52478 (red) and DAPI (blue).

For double immunostaining, sections were first stained overnight at 4°C with anti-Neu5Gc after blocking in 10% (Neu5Gc-free) human serum, after blocking in 5 mg/mL BSA, sections were incubated overnight with both primary antibodies without fixation, washed for one hour and incubated with the appropriate secondary antibodies.

IHC image of CD11b staining in a formalin fixed, paraffin embedded normal human 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 (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with unpurified ab52478, 5 µg/ml, for 15 minutes 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

Immunohistochemical analysis of paraffin-embedded human spleen tissue using unpurified ab52478 at a dilution of 1/100.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.