Figure 1: F4/80 target protein structure.

F4/80 Target Introduction

Protein Function

• F4/80 is an orphan receptor involved in cell adhesion and may be involved in cell-cell interactions related explicitly to immune system cells. It may also play a role in developing regulatory T cells (Treg).
• It belongs to the G protein-coupled receptor 2 family, the adhesion G protein-coupled receptor (ADGR) subfamily.

Protein Expression

• F4/80 is expressed in various mature macrophages.
• The expression of mouse-derived F4/80 varies among different populations of mouse monocytes, with expression in various mature macrophages such as Kupffer cells in the liver, Langerhans cells in the skin, microglia in the brain, and macrophages in the peritoneum, intestinal lamina propria, splenic red pulp, lung, thymus, and bone marrow matrix. Expression is very low or absent in osteoclasts, macrophages in the T cell zone and marginal zone, alveolar macrophages, and most classical dendritic cells.
• Human-derived F4/80 is only expressed in eosinophils. In addition to eosinophils, human-derived F4/80 can also be expressed in gastrointestinal macrophages and liver Kupffer cells. F4/80 can also be detected in respiratory epithelial cells in the nasopharynx and bronchi.

Protein Localization

• F4/80 is mainly localized to the cell membrane, with some also localized to the cytoplasm.

Isoforms & Post-translational Modifications

• Human (Q14246): Isoforms 1-3: 90-95 kDa (predicted)
• Isoforms 4-5: 79-83 kDa (predicted)
• Mouse (Q61549): Isoform 1: 102 kDa (predicted)
• Rat (Q5Y4N8): Isoform 1: 102 kDa (predicted)
• F4/80 protein has multiple glycosylation sites.

IHC experiment tips

Precautions

• Mouse-derived F4/80 is mainly expressed on the surface of macrophages and is used as a marker for mature macrophage tissue. It should be noted that macrophage types vary in different tissues, and the expression levels of F4/80 also vary in different subtypes of macrophages. Typically, positive staining of F4/80 can be observed in Kupffer cells in the liver, red pulp macrophages in the spleen, microglia in the brain, as well as resident macrophages in the bone marrow stroma, lamina propria, kidney, lymph nodes, and peritoneum. If the positive signal proportion is too low or no signal is detected during microscopy, we recommend changing the section to find a more suitable cell type and field of view for observation.
• When analyzing the subcellular localization of F4/80, please note that F4/80 may be located not only on the cell membrane but also in the cytoplasm, so cytoplasmic staining can be explained.

Positive controls

• Mouse liver and spleen tissue
• Human colon tissue
• M1 and M2 macrophages derived from mouse colon tissue

Example of results

Figure 2: IHC - Recombinant Anti-F4/80 antibody [EPR26545-166] (ab300421)

Sample name: Mouse liver tissue paraffin section fixed with PFA

Primary antibody:  Dilution 1/5000
Secondary antibody: LeicaDS9800 (Bond™ Polymer Refine Detection)

Antigen retrieval method: Heat-induced antigen retrieval using Tris/EDTA pH 9.0 buffer for 20 minutes

Experimental result: Positive staining of Kupffer cells in mouse liver.

Key control points

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

Sample fixation:

1. The sample fixation time 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 appropriate.
2. Excessive fixation will block antigenic epitopes. Although antigen retrieval will expose some of the epitopes, if the tissue fixation time is very long (such as more than a week), there may still be no signal after antigen retrieval.

Antigen retrieval:

1. When performing immunohistochemistry experiments 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.

Blocking:

1. If HRP conjugates will be used for detection, please use 3% hydrogen peroxide to treat the sections for 10 minutes to block endogenous peroxidase.
2. If fluorescently labeled secondary antibodies are used for the experiment, we recommend using a blocking solution containing 1% BSA and a final concentration of 0.3 M glycine to quench the autofluorescence caused by aldehyde groups.
3. Before incubating with the primary antibody, it is necessary to block with serum and avoid using blocking solutions from the same species as the host. The source of the serum can be selected based on the host of the secondary antibody, for example, if the secondary antibody is Goat Anti-Rabbit IgG H&L (HRP polymer) or Goat Anti-Mouse IgG H&L (HRP polymer), goat serum can be used as the blocking solution.

Antibody incubation:

1. We recommend incubating the antibodies in a humid chamber to prevent drying of the sections.
2. When conducting the initial experiment, please choose the appropriate working concentration of the antibody according to the product manual.
3. Use a signal amplification system to obtain a stronger experimental signal, such as Polymer-conjugated HRP secondary antibodies.

ICC experimental tips

Precautions

• Although F4/80 is widely present in macrophages derived from mice, the expression level of this protein can be influenced by the maturity of the cells, developmental stage, and macrophage subtypes. Before performing the detection, it is necessary to confirm that the macrophages have reached the mature stage and select a subtype of macrophages with sufficient data support for expressing F4/80, such as mature Raw264.7 cells, as a positive cell control, and conduct the experiment together.
• When analyzing the subcellular localization of F4/80, please note that besides being mainly located on the cell membrane, F4/80 may also be located in the cytoplasm, so the occurrence of cytoplasmic staining can be explained (Figure 3).

Positive control

• Raw264.7 cells, mouse macrophages

Example of results

Figure 3: ICC - Recombinant Anti-F4/80 antibody [EPR26545-166] (ab300421)

Sample name: Raw264.7 cells
Permeabilization: 0.1% TritonX-100

Primary antibody: diluted at a concentration of 1/50
Secondary antibody: Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081)

Experimental result:  Positive staining was observed in the cell membrane and cytoplasm of Raw264.7 cells.

Key control points

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

Cell culture:

1. Cell culture is the basis for subsequent ICC detection. Before the experiment, it is necessary to ensure that the cell status is normal. A confluence of 60%-80% is suitable for most samples.

Sample fixation:

1. If aldehydes (such as 4% PFA) are used for fixation, we recommend fixing at room temperature for 10 minutes.
2. If methanol is chosen as the fixative, suspended cells are recommended to be fixed with 80% methanol for 5 minutes, and adherent cells are recommended to be fixed with 100% methanol for 5 minutes.
3. Do not over-fix the sample, as it will reduce the signal.

Permeabilization:

1. If cells are fixed with aldehydes (such as 4% PFA), it is recommended to use 0.1-0.25% Triton-X 100 for room temperature permeabilization for 10 minutes.

Blocking:

1. We recommend using PBS containing 0.1% tween20, 1% BSA, 10% serum from the secondary antibody source, and 0.3 M glycine to quench the autofluorescence caused by aldehyde groups.
   Add 0.3 M glycine to the blocking solution to quench the autofluorescence caused by aldehyde groups.

Antibody incubation:

1. When conducting the initial experiment, please refer to the specific antibody's instructions to choose the appropriate antibody working concentration.
2. We recommend incubating the antibodies in a humid chamber to avoid drying out.
3. We recommend incubating the primary antibody overnight at 4°C to obtain a stronger signal and lower background.
4. Please incubate the fluorescently labeled secondary antibody in the dark to avoid photobleaching.

References

1. J M Austyn, S Gordon. F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur J Immunol. 1981 Oct;11(10): 805-15.doi:10.1002/eji.1830111013.
2. Lindsey A Waddell, Lucas Lefevre, Stephen J Bush, etc. ADGRE1 (EMR1, F4/80) Is a Rapidly-Evolving Gene Expressed in Mammalian Monocyte-Macrophages. Front Immunol. 2018 Oct 1;9:2246. doi: 10.3389/fimmu.2018.02246. eCollection 2018.
3. Jörg Hamann, Nathalie Koning, Walter Pouwels, etc. EMR1, the human homolog of F4/80, is an eosinophil-specific receptor. Eur J Immunol. 2007 Oct;37(10):2797-802. doi: 10.1002/eji.200737553.
4. V Baud, S L Chissoe, E Viegas-Péquignot, S Diriong, V C N'Guyen, B A Roe, M Lipinski. EMR1, an unusual member in the family of hormone receptors with seven transmembrane segments. Genomics. 1995 Mar 20;26(2):334-44. doi: 10.1016/0888-7543(95)80218-b.