**4. Practical issues involved in microglial phenotyping in human autopsy brains**

activated to be more or less efficient at Aβ removal [30, 31]. This was particularly shown in Aβ-peptide immunized mice, which had produced a specific antibody response to plaque material. The coating of plaques with anti-Aβ immunoglobulin appeared to promote phagocytosis through engagement of the microglial IgG Fc receptors. Overall, these studies showed that microglia of a particular phenotype have the potential to remove Aβ; similar observations have come from human pathological studies in certain subjects who had received the

**3. Schemes for defining microglial function: limitations of M1 and** 

Phenotyping of macrophages and microglia progressed with the pioneering work of Gordon and colleagues who sought to develop schemes for classification of macrophages, first in mice and then in humans, by assigning activation states to the expression of different antigenic markers based on responses to defined activation stimuli [32, 33]. Much of this work employed gene expression profiling mRNA analysis since these techniques have fewer of the limitations associated with antibodies that will be discussed. These phenotyping schemes were also applied to microglia, both rodent and human. The scheme defined classical activation or M1 activation, as being the state of macrophages/microglia that have been stimulated with strong inflammatory agents such as lipopolysaccharide (LPS) and interferon gamma (IFN-γ). Such activated cells will be expressing increased levels of cytokines and enzymes such as TNF-α, IL-1β, IL6, matrix metalloproteinase (MMP)-3 and MMP-9. The corollary to this is alternative activation or M2, which defines the markers and products of cells responding to anti-inflammatory cytokines such as IL-4 or IL-13. These cells have a reparative/neurotrophic phenotype and can produce growth factors. Such reparative M2 microglia also show increased phagocytosis. The M2 scheme was further subdivided into M2a (responses to IL4 or IL13), M2b (responses to immune complexes in combination with IL-1β or LPS) and M2c (responses to anti-inflammatory IL-10, TGFβ or glucocorticoids). It was shown that increased expression of the scavenger receptor CD163, a marker for M2c was upregulated in microglia in AD and Parkinson disease dementia cases. This is the first study showing a type of alterna-

Many studies have tried to apply these schemes to tissue microglia but their validity has been contested [35]. The schemes are dependent on using defined stimuli, while in the degenerating AD brain, there will be many different stimuli (Aβ and tau in different conformations, reactive oxygen, cytokines, bioactive lipids, ATP/ADP, DNA, etc.) that will account for the heterogeneity of microglia responses in tissue. In recent years, there has been criticism that the M1 and M2 scheme is not applicable for tissue microglia as such defined microglia do not seem to exist in brain [35]. This may be correct as the microenvironment around every plaque and every neuron will be different, but to attempt to profile microglia does require some form of scheme, even an imperfect one, to relate to function. It will also be proposed that the limitations of the M1 and M2 classification schemes could be due to technical reasons as much as

Aβ vaccine [14].

biological reasons.

**M2 activation state definitions**

44 Alzheimer's Disease - The 21st Century Challenge

tive activation in AD tissues by immunohistochemistry [34].

Success in classifying microglia in postmortem human autopsy tissue sections is primarily dependent on the antibodies being used for this purpose, but also the manner in which the brain tissue being studied was preserved. Many published studies of microglial markers for immunohistochemistry have been restricted to antibodies that produce strong immunoreactivity on extensively fixed tissue sections. This is particularly true for HLA-DR, which is the most widely used for human microglial studies, as available antibodies can produce vivid results on a wide range of preserved brain tissue. The following references are the first for HLA-DR and the most recent, spanning 30 years of studies [36, 37]. The function of HLA-DR in AD microglia is still unclear. This protein functions to present processed antigens to T lymphocytes that are not present in the AD microenvironment. The signaling that leads to upregulation of HLA-DR in AD microglia has not been defined. In recent years, the marker IBA-1, which recognizes an actin-binding protein involved in cytoskeletal reorganization and cell motility, has also been extensively used to identify microglia because of the availability of robust staining antibodies [38]. IBA-1 antibodies seem to recognize all microglia with limited upregulation in activated microglia, though this interpretation is also dependent on observations related to microglial morphology. The use of antibodies that produce strong results in tissue sections may have biased our understanding of microglial function in disease as many other antigenic markers are present, but suitable antibodies to reveal them in tissue are not available. Most useful markers of function are cell-surface glycosylated proteins whose antigenicity become significantly affected by fixation conditions and also by the degree of glycosylation. The most widely available tissues for many researchers are tissue blocks that have been formalin-fixed for extended periods and then embedded in paraffin (FFPE); this process includes treatments with xylene. These preservation methods strongly affect the ability of antibodies to recognize many antigens, but in particular cell-surface glycoproteins. The numbers of antibodies that are effective at antigen recognition on FFPE tissue are a small percentage of available antibodies. In addition, the use of FFPE tissue usually requires the application of antigen retrieval techniques for most antibodies to work; there are a range of these methods but their successful application is dependent on operator skill and can lack reproducibility. As mentioned, the applicability of M1 and M2-like schemes to classify microglia in human brain samples has been criticized as many of the classification antigens have not been proven in tissue microglia [35], however such schemes may have been prematurely discarded due to the lack of panels of antibodies functional on available brain tissue samples.

#### **4.1. Previous studies of microglial functional proteins in AD**

Since the initial studies of increased HLA-DR expression by microglia in AD brains, in areas associated with pathology [9, 19, 36, 39–42], expression of a range of macrophage markers have been applied to AD brain tissues. These include beta II integrins (CD11a, b, and c and CD18 complement and phagocytic receptors), immunoglobulin Fc receptors (CD16, CD32, CD64) [11], lipopolysaccharide receptor CD14 [43], macrophage colony stimulating factor receptor-1 (CSF-1R; CD115) [44], type B scavenger receptor CD36 [45, 46], ferritin [47], signal regulatory protein beta-1 (SIRPβ-1) [48] and progranulin [49]. The markers CD43 and TMEM106B were shown to be downregulated in AD microglia compared to controls [50, 51]. This represents an incomplete list due to space limitations but many of these markers are related to phagocytic function rather than cytotoxicity. Ferritin has unique properties in relation to microglial activation as it is a ubiquitous iron transport protein but in tissue seems to selectively identify a population of activated microglia [12, 13]. To directly demonstrate potential cytotoxicity, the demonstration of increased levels of cytokines in microglia is needed. Over the last 30 years, there have been few studies using immunohistochemistry to profile cytokines in tissue sections. A series of studies by Griffin and colleagues showed IL-1α-expressing microglia were associated with different types of plaques and tangles. Diffuse neuritic and non-neuritic plaques had the most IL-1α positive microglia, while dense core neuritic and non-neuritic plaques had significantly few IL-1α positive microglia. These results suggest that this population of microglia were involved at early stages of plaque formation [52]. Use of this marker demonstrated that IL-1α positive microglia were involved in the generation of neurofibrillary tangles in the parahippocampal gyrus [53]. In another study, it was shown that IL-1β and TNF-α could be localized to microglia in human AD tissue [42]. The limited numbers of studies do highlight the technical difficulties of detecting secreted proteins such as cytokines. Griffin and colleagues employed FFPE tissue for immunohistochemistry. We have attempted a number of times using our short-fixed microtome cut sections to localize cytokines to tissue and have never been successful. As these molecules are secreted rather than membrane localized, it is possible the hard fixation involved in FFPE is needed to anchor them, and then antigen retrieval to allow antibody access. With short fixed brain tissues materials, these soluble proteins might not be adequately fixed *in situ* for localization.

to what might be expected in human tissues. In addition, the immunizing proteins usually cover the majority of the native protein, and thus preserve some of the secondary protein structure that affects antigenicity, along with containing multiple antigenic epitopes. These proteins produce antibodies with immunogenicity superior to the strategy of many companies that use short synthetic peptide sequences of 10–20 amino acids as immunogens, and then conjugated to a carrier prior to animal immunization. Our experience with R & D Systems affinity purified polyclonal antibodies has generally been favorable for use on lightly-fixed tissues. These antibodies will contain a range of epitopes that can increase the likelihood of identifying epitopes on proteins not severely affected by fixation. The use of large protein antigens to prepare polyclonal does have some drawbacks as there is the potential for cross reactivity with other related proteins. Quality control is dependent on being able to carry out protein absorption of antibody to show removal of tissue reactivity, along with western blot

Defining Microglial Phenotypes in Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.75511 47

**Figure 2** (panels A and B) illustrates our experience using an R & D Systems antibody to Toll-like receptor (TLR)-3 (AF1487) to identify microglia in AD brains, and an R & D Systems polyclonal antibody to CD206 (AF2535), which failed to identify microglia (panels D and E). The TLR-3 polyclonal antibody could identify structures in human brain microglia (**Figure 2**, panels A and B). One comment is that if western blots are carried out using complex material such as brain material, the presence of other protein bands, besides the full length protein should be anticipated (**Figure 2**, panel C). Most functional proteins are either cleaved during their normal function, for example loss of leader sequences, cleaved to produce secreted forms, or broken down as part of cellular metabolism. Interestingly, a monoclonal antibody to TLR-3 produced with the same immunizing protein could not stain microglia in tissue, but this antibody will be specific for only a single epitope present in the immunizing protein. We have had similar experience with an R &D systems antibody to CD206 (**Figure 2**, panels D and E), also known as macrophage mannose receptor, produced against a eukaryotic cell expressed protein. This protein has been defined as a prototypical marker for M2a alternative activation as its expression is induced in the presence of IL-4. We used this antibody to determine if there was evidence for alternative activation microglia in human brains. Using this antibody, which on western blots could detect specific bands on brain samples, did not identify microglia in any of the control or AD tissue sections we stained. Noticeable however was the strong CD206 staining of round cells (perivascular macrophages) located within or around the vessels present in the brain sections. This is similar to a published finding [34]. This seems to indicate alternative activated macrophages are common in vessels, while alternative activated microglia are not present in neuropil. In human brains, identifying expression of inflammatory associated molecules at the RNA or protein level using brain homogenates need to be interpreted with caution as significant numbers of blood cells can be trapped within the tissue [57]. Confirmation of findings by immunohistochemistry is needed when making observations relevant to microglia. The absence of alternative activated markers in AD brain samples was confirmed for the CD200 receptor (CD200R). This is a myeloid specific receptor that is activated by the ligand CD200 to induce anti-inflammatory signaling. We showed that it was induced by IL-4 and IL-13 and fit the classical definition of an M2a marker, similar to CD206. Using several antibodies, including R&D Systems polyclonal antibody (AF3414) and a custom peptide antibody, we could not localize CD200R

detection of specific protein bands.

#### **4.2. Selecting antibodies for microglial phenotyping**

The whole field of human brain immunohistochemistry has several limitations when it comes to selection of suitable antibodies needed to reveal location of proteins of interest. Firstly, the antibody, usually a monoclonal antibody of mouse or rabbit origin, thus specific to an epitope representing a small portion of the target protein, has to be able to show specificity—namely it is actually recognizing the target protein *in situ* and not cross reacting with other proteins. Secondly, the antibody, if it can be validated to recognize the target protein in tissue, its specificity and sensitivity can be affected by the fixation conditions. In our experience, the study of microglial antigens with a wider range of antibodies has been less problematic using brain tissue fixed for a short period (48 h) in paraformaldehyde (not formalin) and then cryoprotected and sectioned using a freezing microtome. This process avoids the harsh treatments involved in paraffin embedding of tissue. Over the years, we have successfully identified microglial proteins CD87 [54], RAGE, CD33 [55], TREM-2 [56], TLR-2, -3, -4 along with HLA-DR, IBA-1, CD68 in AD tissues.

Our experience with antibodies when using these tissues identified some features that help increase the chances of successful immunolocalization. One company—R & D Systems— Biotechne, Minneapolis, MN—have produced many of their antibodies using a relatively unique strategy for the industry. Many of their antibodies were prepared from proteins of interest expressed in eukaryotic cells. These proteins will be glycosylated in a similar manner to what might be expected in human tissues. In addition, the immunizing proteins usually cover the majority of the native protein, and thus preserve some of the secondary protein structure that affects antigenicity, along with containing multiple antigenic epitopes. These proteins produce antibodies with immunogenicity superior to the strategy of many companies that use short synthetic peptide sequences of 10–20 amino acids as immunogens, and then conjugated to a carrier prior to animal immunization. Our experience with R & D Systems affinity purified polyclonal antibodies has generally been favorable for use on lightly-fixed tissues. These antibodies will contain a range of epitopes that can increase the likelihood of identifying epitopes on proteins not severely affected by fixation. The use of large protein antigens to prepare polyclonal does have some drawbacks as there is the potential for cross reactivity with other related proteins. Quality control is dependent on being able to carry out protein absorption of antibody to show removal of tissue reactivity, along with western blot detection of specific protein bands.

protein beta-1 (SIRPβ-1) [48] and progranulin [49]. The markers CD43 and TMEM106B were shown to be downregulated in AD microglia compared to controls [50, 51]. This represents an incomplete list due to space limitations but many of these markers are related to phagocytic function rather than cytotoxicity. Ferritin has unique properties in relation to microglial activation as it is a ubiquitous iron transport protein but in tissue seems to selectively identify a population of activated microglia [12, 13]. To directly demonstrate potential cytotoxicity, the demonstration of increased levels of cytokines in microglia is needed. Over the last 30 years, there have been few studies using immunohistochemistry to profile cytokines in tissue sections. A series of studies by Griffin and colleagues showed IL-1α-expressing microglia were associated with different types of plaques and tangles. Diffuse neuritic and non-neuritic plaques had the most IL-1α positive microglia, while dense core neuritic and non-neuritic plaques had significantly few IL-1α positive microglia. These results suggest that this population of microglia were involved at early stages of plaque formation [52]. Use of this marker demonstrated that IL-1α positive microglia were involved in the generation of neurofibrillary tangles in the parahippocampal gyrus [53]. In another study, it was shown that IL-1β and TNF-α could be localized to microglia in human AD tissue [42]. The limited numbers of studies do highlight the technical difficulties of detecting secreted proteins such as cytokines. Griffin and colleagues employed FFPE tissue for immunohistochemistry. We have attempted a number of times using our short-fixed microtome cut sections to localize cytokines to tissue and have never been successful. As these molecules are secreted rather than membrane localized, it is possible the hard fixation involved in FFPE is needed to anchor them, and then antigen retrieval to allow antibody access. With short fixed brain tissues materials, these soluble

The whole field of human brain immunohistochemistry has several limitations when it comes to selection of suitable antibodies needed to reveal location of proteins of interest. Firstly, the antibody, usually a monoclonal antibody of mouse or rabbit origin, thus specific to an epitope representing a small portion of the target protein, has to be able to show specificity—namely it is actually recognizing the target protein *in situ* and not cross reacting with other proteins. Secondly, the antibody, if it can be validated to recognize the target protein in tissue, its specificity and sensitivity can be affected by the fixation conditions. In our experience, the study of microglial antigens with a wider range of antibodies has been less problematic using brain tissue fixed for a short period (48 h) in paraformaldehyde (not formalin) and then cryoprotected and sectioned using a freezing microtome. This process avoids the harsh treatments involved in paraffin embedding of tissue. Over the years, we have successfully identified microglial proteins CD87 [54], RAGE, CD33 [55], TREM-2 [56], TLR-2, -3, -4 along with HLA-DR, IBA-1,

Our experience with antibodies when using these tissues identified some features that help increase the chances of successful immunolocalization. One company—R & D Systems— Biotechne, Minneapolis, MN—have produced many of their antibodies using a relatively unique strategy for the industry. Many of their antibodies were prepared from proteins of interest expressed in eukaryotic cells. These proteins will be glycosylated in a similar manner

proteins might not be adequately fixed *in situ* for localization.

**4.2. Selecting antibodies for microglial phenotyping**

46 Alzheimer's Disease - The 21st Century Challenge

CD68 in AD tissues.

**Figure 2** (panels A and B) illustrates our experience using an R & D Systems antibody to Toll-like receptor (TLR)-3 (AF1487) to identify microglia in AD brains, and an R & D Systems polyclonal antibody to CD206 (AF2535), which failed to identify microglia (panels D and E). The TLR-3 polyclonal antibody could identify structures in human brain microglia (**Figure 2**, panels A and B). One comment is that if western blots are carried out using complex material such as brain material, the presence of other protein bands, besides the full length protein should be anticipated (**Figure 2**, panel C). Most functional proteins are either cleaved during their normal function, for example loss of leader sequences, cleaved to produce secreted forms, or broken down as part of cellular metabolism. Interestingly, a monoclonal antibody to TLR-3 produced with the same immunizing protein could not stain microglia in tissue, but this antibody will be specific for only a single epitope present in the immunizing protein. We have had similar experience with an R &D systems antibody to CD206 (**Figure 2**, panels D and E), also known as macrophage mannose receptor, produced against a eukaryotic cell expressed protein. This protein has been defined as a prototypical marker for M2a alternative activation as its expression is induced in the presence of IL-4. We used this antibody to determine if there was evidence for alternative activation microglia in human brains. Using this antibody, which on western blots could detect specific bands on brain samples, did not identify microglia in any of the control or AD tissue sections we stained. Noticeable however was the strong CD206 staining of round cells (perivascular macrophages) located within or around the vessels present in the brain sections. This is similar to a published finding [34]. This seems to indicate alternative activated macrophages are common in vessels, while alternative activated microglia are not present in neuropil. In human brains, identifying expression of inflammatory associated molecules at the RNA or protein level using brain homogenates need to be interpreted with caution as significant numbers of blood cells can be trapped within the tissue [57]. Confirmation of findings by immunohistochemistry is needed when making observations relevant to microglia. The absence of alternative activated markers in AD brain samples was confirmed for the CD200 receptor (CD200R). This is a myeloid specific receptor that is activated by the ligand CD200 to induce anti-inflammatory signaling. We showed that it was induced by IL-4 and IL-13 and fit the classical definition of an M2a marker, similar to CD206. Using several antibodies, including R&D Systems polyclonal antibody (AF3414) and a custom peptide antibody, we could not localize CD200R

associated with proinflammatory activation. There has only been a single study describing microglial immunohistochemistry in human AD brains for CD14 [43]. Using short fixation tissue, we reexamined expression of this marker (**Figure 2**, panels F and G). It is strongly expressed by most vascular macrophages of all cases (**Figure 2**, panel F—ND case), but increased expression was readily detectable in subsets of IBA-1 microglia in AD cases (**Figure 2**, panel G—purple). As CD14 can bind Aβ with proinflammatory activation through interaction with TLR-2 or TLR-4, increased CD14 expression could be a more used marker for defining

Defining Microglial Phenotypes in Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.75511 49

This discussion is of particular relevance for considering microglial phenotyping of TREM-2 positive microglia. Considerable interest in the role of TREM-2 in AD has spurred new interest in neuroinflammation and AD. A single nucleotide polymorphism (SNP) in the TREM-2 gene (rs75932628) that results in a mutation in the TREM-2 protein (R47H) can increase the risk of developing AD by 2- to 11-fold depending on the population studied [59, 60]. Mutations in TREM-2 or its adaptor protein DAP12 were first identified in Nasu-Hakola disease, which leads to early onset dementia amongst other symptoms [61]. The mutation appears to lead to loss of function of the TREM-2 protein, whose normal function is to promote phagocytosis of apoptotic neurons through binding to heat shock protein 70 (hsp70) or different conformations of lipids. There have been few studies of immunohistochemistry of TREM-2 in human AD brains, which appears mainly due to lack of robust antibodies for pathological work. We published one of the first studies that showed plaque- and tangle-associated microglia were positive for TREM-2 [56]. In this study, we had to screen a number of antibodies for specificity and sensitivity in human brain tissue. The best results were obtained with an R&D Systems polyclonal antibody to TREM-2 (AF1828) prepared using a eukaryotic cell expressed protein corresponding to 75% of the protein and to the complete extracellular domain. A recent study of TREM-2 expression in AD frontal cortex using an antibody prepared using a peptide corresponding to N-terminal amino acids 29–59 of human TREM2 (ab175262, Abcam, Cambridge, MA) showed specificity by western blots, but these authors presented no data on TREM2 immunohistochemistry [62]. TREM-2 expression is restricted to dendritic/myeloid cells and is high in brain microglia. Specificity of commercial antibodies has been an issue, but also the sensitivity of detection. Two studies have concluded that TREM-2 was not expressed by microglia in brain, but both studies employed FFPE tissue samples with antigen retrieval [63, 64]. One study showed that the R&D antibody was specific for TREM-2, similar to our published work, but they could not demonstrate microglial TREM-2 immunoreactivity [63]. Similar to our previous studies, we employed lightly fixed brain tissues that were not paraffin-embedded [56]. With these sections, we could demonstrate specific TREM2 localization to microglia [56]. Our finding is reasonable as TREM-2

has been localized to plaque-associated microglia in AD model transgenic mice [65].

**5. Does expression of antigen correlate with identifiable function**

How does antigen expression relate to demonstrated microglial function? With the exception of HLA-DR and IBA-1, most studies of microglia in human brains have not been adequately

cytotoxic microglia.

**4.3. Profiling TREM-2 microglia in human brains**

**Figure 2.** Approaches to microglial phenotyping in Alzheimer's disease brains. (A) and (B) Immunohistochemistry for a new marker for microglia (toll-like receptor-3: TLR-3 in human brains. Double immunostaining for TLR-3 (purple) colocalizing with IBA-1 microglia in (A) non-demented control middle temporal gyrus and (B) Alzheimer's disease case. See text for further explanation. These findings were obtained using R&D Systems antibody (AF1868). (C) Western blot of human brain samples for TLR-3. This panel illustrates that protein bands other than full length peptides can be present in biological samples. (D) and (E) Absence of alternatively activated microglia expressing CD206 in ND (D) or AD (E) temporal cortex brain sections but positive expression in perivascular/vascular macrophages. (F) and (G) The proinflammatory marker CD14 does show increased expression by microglia in AD cases (purple) colocalizing with IBA-1 immunoreactivity brain Strong positive staining is present in perivascular/vascular macrophages (purple) is also a feature.

immunoreactivity to brain microglia even though protein and mRNA expression of CD200R are detectable in human brains [58].

One marker that seems to have been overlooked in microglial profiling in tissue is CD14, the LPS co- receptor. This receptor is a classical M1-like activation marker with upregulation associated with proinflammatory activation. There has only been a single study describing microglial immunohistochemistry in human AD brains for CD14 [43]. Using short fixation tissue, we reexamined expression of this marker (**Figure 2**, panels F and G). It is strongly expressed by most vascular macrophages of all cases (**Figure 2**, panel F—ND case), but increased expression was readily detectable in subsets of IBA-1 microglia in AD cases (**Figure 2**, panel G—purple). As CD14 can bind Aβ with proinflammatory activation through interaction with TLR-2 or TLR-4, increased CD14 expression could be a more used marker for defining cytotoxic microglia.

#### **4.3. Profiling TREM-2 microglia in human brains**

This discussion is of particular relevance for considering microglial phenotyping of TREM-2 positive microglia. Considerable interest in the role of TREM-2 in AD has spurred new interest in neuroinflammation and AD. A single nucleotide polymorphism (SNP) in the TREM-2 gene (rs75932628) that results in a mutation in the TREM-2 protein (R47H) can increase the risk of developing AD by 2- to 11-fold depending on the population studied [59, 60]. Mutations in TREM-2 or its adaptor protein DAP12 were first identified in Nasu-Hakola disease, which leads to early onset dementia amongst other symptoms [61]. The mutation appears to lead to loss of function of the TREM-2 protein, whose normal function is to promote phagocytosis of apoptotic neurons through binding to heat shock protein 70 (hsp70) or different conformations of lipids. There have been few studies of immunohistochemistry of TREM-2 in human AD brains, which appears mainly due to lack of robust antibodies for pathological work. We published one of the first studies that showed plaque- and tangle-associated microglia were positive for TREM-2 [56]. In this study, we had to screen a number of antibodies for specificity and sensitivity in human brain tissue. The best results were obtained with an R&D Systems polyclonal antibody to TREM-2 (AF1828) prepared using a eukaryotic cell expressed protein corresponding to 75% of the protein and to the complete extracellular domain. A recent study of TREM-2 expression in AD frontal cortex using an antibody prepared using a peptide corresponding to N-terminal amino acids 29–59 of human TREM2 (ab175262, Abcam, Cambridge, MA) showed specificity by western blots, but these authors presented no data on TREM2 immunohistochemistry [62]. TREM-2 expression is restricted to dendritic/myeloid cells and is high in brain microglia. Specificity of commercial antibodies has been an issue, but also the sensitivity of detection. Two studies have concluded that TREM-2 was not expressed by microglia in brain, but both studies employed FFPE tissue samples with antigen retrieval [63, 64]. One study showed that the R&D antibody was specific for TREM-2, similar to our published work, but they could not demonstrate microglial TREM-2 immunoreactivity [63]. Similar to our previous studies, we employed lightly fixed brain tissues that were not paraffin-embedded [56]. With these sections, we could demonstrate specific TREM2 localization to microglia [56]. Our finding is reasonable as TREM-2 has been localized to plaque-associated microglia in AD model transgenic mice [65].
