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Macrophages are of special interest as they play essential roles in immunity, inflammation, and tissue homeostasis, and their activity is relevant across a range of diseases, including cancer, autoimmune disorders, and infectious diseases. Multiplex immunohistochemistry (IHC) enables the simultaneous visualization of multiple markers in the same tissue section, providing insight into the complexity of the immune landscapes in tissues. An important marker for identifying macrophages in mice is F4/80, an established macrophage-specific antigen that is frequently used to label and characterize these cells.
F4/80, also known as EMR1 or ADGRE1, belongs to the epidermal growth factor (EGF)-transmembrane 7 (TM7) family, which is part of the G protein-coupled receptor (GPCR) superfamily (Baud et al., 1995). However, its expression can vary depending on the type of macrophage and its state of maturation or activation. For example, F4/80 is highly and constitutively expressed on yolk sac-derived tissue-resident macrophages in mice, including Kupffer cells in the liver, Langerhans cells of the skin, microglia in the brain or red pulp macrophages in the spleen (Figure 1).
Other macrophages that express somewhat low levels of F4/80 are presumed to be of hematopoietic origin (see: Mouse Tissue-resident Macrophages). The tissue-specific expression of F4/80 suggests that it may have unique roles depending on the macrophage population. The function of F4/80 has been a subject of significant interest, particularly in relation to its role in immune responses, tissue homeostasis, and inflammation. F4/80-positive macrophages induce the development of antigen-specific efferent regulatory T cells (T regs) and thus play a major role in the regulation of immune tolerance (Lin et al., 2005).
In addition to its expression on macrophages, F4/80 is also found on a subset of dendritic cells (DCs), eosinophils, and certain monocyte populations (Garry et al., 1991, Gautier et al., 2012). Nevertheless, F4/80 is a better macrophage marker than other anti-macrophage antibodies in mice (e.g. CD68, Mac-1, Mac-2, Mac-3), as F4/80 clearly distinguishes macrophages from fibroblasts (Inoue et al., 2005). Since DCs do not express MerTK and FcγRI, these markers can be combined with F4/80 to distinguish macrophages and DCs (Gautier et al., 2012). Other markers can be combined with F4/80 to recognize macrophage subsets (e.g. CD169, MARCO) or activation states (e.g. iNOS, Arg-1).
Using F4/80 in multiplex IHC offers several benefits for studying macrophages in tissues, but there are also challenges to consider. Highly specific F4/80 antibodies are needed that bind macrophages, but not closely related hematopoietic cells in complex tissue environments. For compatibility with other markers, it is advantageous if the antibodies work in a variety of tissue-based methods and protocols. Therefore, we investigated the staining properties of SYSY|HistoSure F4/80 antibodies in different IHC protocols in formalin-fixed tissue (IHC-P), as well as using different post-fixation methods in fresh frozen tissue.
In IHC-P, the staining properties of the antibodies (e.g. sensitivity, specificity, signal-to-noise ratio) are often influenced by the pH of the buffer used for heat-induced antigen (epitope) retrieval (HIER). To facilitate combination with other antibodies in multiple staining, antibodies that function robustly in different protocols are required.
We therefore compared our rat anti-F4/80 monoclonal antibody HS-397 017 (rt mAb) and its recombinant rabbit derivative HS-397 008 (rb mAb) with frequently used external antibodies (clone Cl:A3-1, rt mAb; clone SP115, rb mAb) in IHC-P on mouse spleen, using different antigen retrieval (AR) buffers (Figure 2). HIER was performed in a vegetable steamer for 30 min, respectively. While the F4/80 clone Cl:A3-1 showed good staining performance only after AR in the pH 6.1 target retrieval solution from Agilent (S1699), the clone SP115 required AR in pH 9 buffer (S2367, Agilent). In contrast, the monoclonal rat F4/80 antibody HS-397 017 and its recombinant rabbit derivative HS-397 008 showed a strong and clean staining independent of the buffer used for AR. This enables general use of SYSY|HistoSure F4/80 antibodies in various IHC-P protocols and allows the combination with a large number of other antibodies for multiplexing.
Figure 2: Anti-F4/80 staining in formalin fixed paraffin embedded (FFPE) mouse spleen sections using different F4/80 antibodies and AR buffers. First row: 10 mM Citrate AR buffer (10 mM citrate, 0.05% Tween-20, pH 6.0). Second row: S1699 Target Retrieval Solution (Agilent, pH 6.1). Third row: S2367 Target Retrieval Solution (Agilent, pH 9.0). First column: F4/80 clone CI:A3-1 (Invitrogen, MA5-16630, 1:50). Second column: F4/80 clone SP115 (Invitrogen, MA5-16363, 1:50). Third column: F4/80 rt mAb HS-397 017 (SYSY|HistoSure, 1:100). Fourth column: F4/80 rb mAb HS-397 008 (SYSY|HistoSure, 1:1000).
Tissue-based detection methods differ in the type of fixation of the tissue used. A distinction is made between pre-fixed tissues and unfixed, freshly frozen tissues, which are usually post-fixed. Therefore, the staining performance of the anti-F4/80 antibody HS-397 017 was analyzed in spleen sections from mice prepared according to the most commonly used tissue preparation protocols. Spleens of mice were either snap frozen or fixed in 4% formaldehyde for 24 hours after tissue removal. Sections of formaldehyde-fixed spleen were prepared using a vibratome. Fresh frozen tissue sections were prepared using a cryostat and post-fixed with formaldehyde or cold methanol prior to IHC staining. Regardless of the tissue preparation or post-fixation method, the anti-F4/80 HS-397 017 antibody showed strong specific staining in red pulp macrophages (Figure 3).
Figure 3: Indirect immunostaining of differently fixed mouse spleen sections with rat anti-F4/80 antibody (cat. HS-397 017, dilution 1:500, red) A: Vibratome section of a mouse spleen fixed in 4% formaldehyde for 24 hours, B: Fresh frozen cryosection, post-fixed for 15 min in 4% formaldehyde, C: Fresh frozen cryosection, post-fixed with cold methanol for 10 minutes.
The mouse spleen can be microanatomically divided into three zones: white medulla, red medulla and the marginal zone (MZ), which are colonized by different macrophage subsets (Da Silva et al., 2015) (Figure 4).
Red pulp macrophages are located in the red medulla and are characterized by high F4/80 and CD68 expression. White pulp macrophages, also known as tingible-body macrophages, express CD68 but lack F4/80. The MZ contains two distinct macrophage populations: the marginal metallophilic macrophages (MMMs), which express CD169 (sialoadhesin), while the macrophages of the outer marginal zone (MZMs) express DC-SIGN/SIGNR1 and the scavenger receptor MARCO (Gordon et al., 2014, Da Silva et al., 2015). In contrast, the protein ionized calcium-binding molecule 1 (IBA1) is expressed by both red and white pulp macrophages (Pislyagin et al., 2017).
The combination of F4/80 with markers specific for different splenic macrophage populations enables their visualization in multiplex IHC. In addition to the rat and recombinant rabbit monoclonal antibodies, SYSY|HistoSure also offers a polyclonal antibody from guinea pig for this purpose (HS-397 004, gp pAb). In multiplex IHC in mouse spleen red pulp macrophages were visualized by co-expression of IBA1 and F4/80 (Figure 5a) or F4/80 and CD68 (Figure 5B). MMMs and MZMs are both F4/80-negative, but express either CD169 (Figure 5C) or MARCO (Figure 5D).
Figure 5: Immunohistochemical doublestaining of mouse spleen sections using different antibody combinations. Nuclei have been visualized by DAPI staining (blue). A: Indirect immunostaining of a PFA fixed mouse spleen section with rabbit anti-IBA1 (cat. no. HS-234 008, dilution 1:250, red) and guinea pig anti-F4/80 (cat. no. HS-397 004, dilution 1:500, green), B: Indirect immunostaining of a FFPE mouse spleen section with rabbit anti-CD68 antibody (cat. no. HS-460 003, dilution 1:1000, red) and rat anti-F4/80 antibody (cat. no. HS-397 017, dilution 1:100, green), C: Indirect immunostaining of a FFPE mouse spleen section with rat anti-CD169 antibody (cat. no. HS-495 017, dilution 1:500, red) and rabbit anti-F4/80 antibody (cat. no. HS-397 008, dilution 1:500, green), D: Indirect immunostaining of a FFPE mouse spleen section using Tyramide signal amplification with rabbit anti-MARCO (cat. no. HS-499 003, 1:200, white) and guinea pig anti-F4/80 (cat. no. HS-397 004, dilution 1:500, green).
The rat monoclonal anti-F4/80 antibody (HS-397 017), its rabbit monoclonal recombinant derivative (HS-397 008) and the polyclonal guinea pig anti-F4/80 antibody (HS-397 004) from SYSY|HistoSure are perfectly suited to visualize macrophage subsets in multiplex IHC:
| Cat. No. | Product Description | Application | Quantity | Price | Cart |
|---|
HS-397 004 | F4/80, Guinea pig, polyclonal, antiserumantiserum | IHC IHC-P | 100 µl | $355.00 |   |
| HS-397 008 | F4/80, rabbit, monoclonal, recombinant IgGrecombinant IgG | WB IHC IHC-P | 100 µl | $420.00 | |
| HS-397 017 | F4/80, rat, monoclonal, purified IgG IgG | WB IHC IHC-P IHC-Fr | 200 µl | $420.00 | |
| HS-397 017BT | F4/80, rat, monoclonal, purified IgG IgG, biotin | WB IHC-P | 100 µg | $470.00 | |
Author: Dr. Christel Bonnas
Scientific Director of HistoSure
Christel has a strong background in immunology and histopathology. She is responsible for antibody development, validation and quality control of our HistoSure product line.
Baud et al., 1995. EMR1, an unusual member in the family of hormone receptors with seven transmembrane segments. PMID: 7601460
Da Silva et al., 2015. Splenic Macrophage Subsets and Their Function during Blood-Borne Infections. PMID: 26441984
Garry et al., 1991. Murine eosinophil granulocytes bind the murine macrophage-monocyte specific monoclonal antibody F4/80. PMID: 1721083
Gautier et al., 2012. Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. PMID: 23023392
Gordon et al., 2014. Macrophage heterogeneity in tissues: phenotypic diversity and functions. PMID: 25319326
Inoue et al., 2005. Antibodies against macrophages that overlap in specificity with fibroblasts. PMID: 15882296
Lin et al., 2005. The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance. PMID: 15883173
Pislyagin et al., 2017. Cucumarioside A₂-2 Causes Macrophage Activation in Mouse Spleen. PMID: 29104230
