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IHC protocol advice:
This antibody is not suitable for IHC on paraffin-embedded samples.
For best results in IHC on frozen tissue, the following may help detection:
1) Paraformaldehyde perfusion fixed samples have worked well for many customers.
2) For non-perfused tissue, either snap freeze or emerse in periodate-lysine-paraformaldehyde (PLP) fixative for 24 hours at 4°C. Reduce the concentration of paraformaldehyde to 0.25-0.5% since this increases the staining intensity for immune cell surface markers (PMID: 7868861).
3) PFA-fixed samples will require cryoprotection by sucrose infiltration.
4) Use OCT (TissueTek) compound for embedding.
5) For snap frozen tissue, fix sections in cold acetone for 10 min. Allow to dry for 10 min at room temperature. Wash with water for 10 min.
6) Do not heat the samples or sections.
7) During the staining procedure, do not allow the sections to dry out.
Our Technical team (firstname.lastname@example.org) will be happy to provide further information and advice.
This antibody clone is manufactured by Abcam.
Our Abpromise guarantee covers the use of ab1211 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-FoFr||Use at an assay dependent concentration.|
|ELISA||1/100 - 1/3200.|
|Flow Cyt||1/25 - 1/200.
(10 µl per million cells).
|IHC-FrFl||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 5 - 10 µg/ml.|
|IP||Use at an assay dependent concentration. PubMed: 3512425|
ab1211 staining CD11b/c (shown in red) in rat spleen tissue sections by IHC (PFA perfusion fixed frozen sections). Tissue samples were fixed with formaldehyde and blocked with BSA for 10 minutes at 21°C. The sample was incubated with primary antibody (1/2000 in TBS/BSA/azide) at 21°C for 16 hours. A Biotin-conjugated Goat anti mouse polyclonal (1/300) was used as the secondary antibody.
NR8383 cells (rat macrophage cell line) stained for CD11b/c (shown in green) using ab1211 in ICC/IF. The cells were fixed with 100% methanol for 10 minutes and then incubated in 1% BSA / 10% normal goat serum / 0.3 M glycine in 0.1% Tween-PBS for 1 hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab1211 at 5µg/ml) overnight at +4°C. The secondary antibody was ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed used at a 1/1000 dilution for 1 hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (shown in red) at a 1/200 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (shown in blue) at a concentration of 1.43 µM for 1 hour at room temperature.
ab1211 staining CD11b/c (shown in green) in rat spleen tissue by IHC (Frozen sections). Tissue was fixed in formaldehyde, blocked with 10% serum for 30 minutes at 24°C, then incubated with ab1211 at a 1/100 dilution. The secondary used was an Alexa-Fluor 488 conjugated goat anti-mouse polyclonal, used at a 1/1000 dilution.
ab1211 staining CD11b/c (shown in red) in rat brain tissue sections by IHC (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with paraformaldehyde and blocked with NGS in 0.3% TritonX-PBS for 60 minutes at 24°C. The sample was incubated with primary antibody (1/300 in NGS in 0.3% TritonX-PBS) at 4°C for 42 hours. Donkey Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (ab150111) was used as the secondary antibody at 1/500.
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