Figure 1: CD11b

CD11b Target Introduction

Protein Function

• CD11b antigen-like family member B (CD11B), also known as integrin alpha M (ITGAM), is involved in various adhesive interactions of monocytes, macrophages, and granulocytes. It regulates neutrophil migration, activation, and apoptosis.
• CD11b in combination with CD45 can be used to distinguish microglia from macrophages. Resting microglia are surface-marked by CD11bhigh and CD45low, while macrophages are CD11bhigh and CD45high.

Protein Expression

• Mainly expressed in monocytes and granulocytes.
• Expressed in neutrophils at the protein level.

Protein Localization

• Localized to the cell membrane.

Figure 2: CD11b ICC experimental result image, Immunocytochemistry/ Immunofluorescence - Anti-CD11b antibody [EPR19387] (ab184308). Green: CD11b; Red: Tubulin; Blue: DAPI.

Isoforms & Post-translational modifications

• Human (P11215): Isoform 1-2: 127 kDa (predicted)
• Mouse (P05555): Isoform 1-2: 115-127 kDa (predicted)
• Presence of disulfide bonds and glycosylation modifications.

IHC experiment tips

Precautions

• The expression level of CD11b may vary in different tissues, and some samples may show weak or no expression (as shown in Figure 3); if you are unsure whether your sample expresses the target protein, we strongly recommend using a positive control in the experiment.

Positive control

• Mouse and human spleen tissues

Example results

Figure 3: IHC- Anti-CD11b antibody [EPR1344] (ab133357).
Tissue microarray staining. This table provides a detailed overview of positive (checkmark) and negative (cross) staining for each sample detection type.

Figure 4: IHC- Anti-CD11b antibody [EPR1344] (ab133357).
Paraffin-embedded human spleen tissue. Primary antibody: ab133357, diluted 1:4000. Secondary antibody: HRP goat anti-rabbit (ab97051). Counterstained with hematoxylin. Antigen retrieval: pH9.0 Tris-EDTA buffer. PBS was used as negative control, as shown in the figure.

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample fixation:

1. The fixation time of the sample depends on the size of the tissue block and the type of tissue, but for most samples, fixing at room temperature for 18-24 hours is more appropriate.
2. Insufficient fixation can result in higher signal at the edges of the sample and weaker signal in the center, or even no signal.
3. Excessive fixation can block antigenic epitopes. Although antigen retrieval can expose some of the epitopes, if the tissue fixation time is very long (more than a week), there may still be no signal after antigen retrieval.
4. We recommend using positive controls.

Antigen retrieval:

1. When performing immunohistochemistry on paraffin sections, we recommend using a pressure cooker for heat-induced antigen retrieval. You can try fixing the sections at 110°C for 15 minutes.
2. When performing immunohistochemistry on frozen sections, organic solvents are usually used for fixation, so antigen retrieval is generally not necessary. If the previous samples were fixed with PFA for 18-24 hours, you can try using a microwave for short-term retrieval or using enzymes for antigen retrieval. However, please optimize the enzyme concentration and retrieval time to avoid damaging the tissue morphology of the sections.

Blocking:

1. If HRP conjugates will be used for detection in subsequent steps, treat the sections with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase.
2. If fluorescently labeled secondary antibodies will be used for the experiment, use 0.3 M glycine during the blocking process to quench the autofluorescence caused by aldehyde groups.

Antibody incubation:

1. Use a signal amplification system to obtain stronger experimental signals, such as Polymer-coupled HRP secondary antibodies.
2. We recommend using fresh antibodies and avoid reusing them.

References

1. R G DiScipio, P J Daffern, I U Schraufstätter, P Sriramarao. Human polymorphonuclear leukocytes adhere to complement factor H through an interaction that involves alphaMbeta2 (CD11b/CD18). J Immunol. 1998 Apr 15;160(8):4057-66.
2. Josephine Losse, Peter F Zipfel, Mihály Józsi. Factor H and factor H-related protein 1 bind to human neutrophils via complement receptor 3, mediate attachment to Candida albicans, and enhance neutrophil antimicrobial activity. J Immunol. 2010 Jan 15;184(2):912-21. doi: 10.4049/jimmunol.0901702. Epub 2009 Dec 11.
3. Ming Bai, Ricardo Grieshaber-Bouyer, Junxia Wang, etc. CD177 modulates human neutrophil migration through activation-mediated integrin and chemoreceptor regulation. Blood. 2017 Nov 9;130(19):2092-2100. doi: 10.1182/blood-2017-03-768507. Epub 2017 Aug 14.
4. Uwe Jerke, Susanne Rolle, Gunnar Dittmar, Behnaz Bayat, Sentot Santoso, Anje Sporbert, Friedrich Luft, Ralph Kettritz. Complement receptor Mac-1 is an adaptor for NB1 (CD177)-mediated PR3-ANCA neutrophil activation. J Biol Chem. 2011 Mar 4;286(9):7070-81. doi: 10.1074/jbc.M110.171256. Epub 2010 Dec 30.
5. H L Pahl, A G Rosmarin, D G Tenen. Characterization of the myeloid-specific CD11b promoter. Blood. 1992 Feb 15;79(4):865-70.
6. Uwe Jerke, Susanne Rolle, Gunnar Dittmar, Behnaz Bayat, Sentot Santoso, Anje Sporbert, Friedrich Luft, Ralph Kettritz. Complement receptor Mac-1 is an adaptor for NB1 (CD177)-mediated PR3-ANCA neutrophil activation. J Biol Chem. 2011 Mar 4;286(9):7070-81. doi: 10.1074/jbc.M110.171256. Epub 2010 Dec 30.