Advances in Visualizing Microglial Cells in Human Central Nervous System Tissue

Abstract

Neuroinflammation has recently been identified as a fundamentally important pathological process in most, if not all, CNS diseases. The main contributor to neuroinflammation is the microglia, which constitute the innate immune response system. Accurate identification of microglia and their reactivity state is therefore essential to further our understanding of CNS pathophysiology. Many staining techniques have been used to visualise microglia in rodent and human tissue, and immunostaining is currently the most frequently used. Historically, identification of microglia was predominantly based on morphological structure, however, recently there has been a reliance on selective antigen expression, and microglia-specific markers have been identified providing increased certainty that the cells observed are in fact microglia, rather than the similar yet distinct macrophages. To date, the most microglia-specific markers are P2Y12 and TMEM119. However, other microglia-related markers can also be useful for demonstrating activation state, phagocytic state, and for neuroimaging purposes in longitudinal studies. Overall, it is important to be aware of the microglia-selectivity issues of the various stains and immunomarkers used by researchers to distinguish microglia in CNS tissue to avoid misinterpretation.

Keywords: immunohistochemistry; immunostaining; microglia; neuroinflammation.

Figure 1

Coexpression of Iba1 and HLA-DR. Human brain biopsy samples from the superior frontal gyrus after TBI were stained with the primary antibodies: rabbit anti-Iba1 (1:1000, Wako, Cat No. 019-19741) and mouse anti-HLA-DR (1:500, Invitrogen, Cat No. MA5-11966) followed by rabbit anti-Alexa Fluor 488 and mouse anti-Alexa Fluor 594, respectively. Limited HLA-DR expression was observed in Iba1+ microglia with a ramified morphology (A–C), but strong HLA-DR expression in Iba1+ microglia in the activated state (D–L) in the non-contused brain tissue. Predominantly amoeboid microglia stained with Iba1 and HLA-DR were observed in contused brain tissue (M–O). Clotted blood from a neurosurgical (brain) operative field was used to identify a few peripherally-derived macrophages that strongly stained for both Iba1 and HLA-DR (P–R). Scale bars are 25 μm. Unpublished data.

Figure 2

Coexpression of Iba1 and CD68. Human brain biopsy samples from the superior frontal gyrus after TBI were stained with the primary antibodies: rabbit anti-Iba1 (1:1000, Wako, Cat No. 019-19741) and mouse anti-CD68 (1:500, Invitrogen, Cat No. 14-0688-82) followed by rabbit anti-Alexa Fluor 488 and mouse anti-Alexa Fluor 594, respectively. Iba1+ microglia co-expressed with CD68 can be observed in various activated states, ranging from the most ramified state (A–C) through increasing states of activation (D–F and G–I) to the most amoeboid morphology (J–L) in the non-contused brain tissue. In contrast, predominantly amoeboid microglia stained with Iba1 and CD68 were observed in contused brain tissue (M–O). Clotted blood from a neurosurgical (brain) operative field was used to identify peripherally-derived macrophages that strongly stained for both Iba1 and CD68 (P–R). Scale bars are 25 μm. Unpublished data.

Figure 3

Coexpression of Iba1 and galectin-3. Human brain biopsy samples from the superior frontal gyrus after TBI were stained with the primary antibodies: rabbit anti-Iba1 (1:1000, Wako, Cat No. 019-19741) and goat anti-galectin-3 (1:500, BioLegend, Cat No. 126701) followed by rabbit anti-Alexa Fluor 488 and goat anti-Alexa Fluor 594, respectively. Limited galectin-3 expression was observed in Iba1+ microglia with a ramified morphology (A–C), but there was a strong galectin-3 expression in Iba1+ microglia in the activated state (D–L) in the non-contused brain tissue. Predominantly amoeboid microglia stained with Iba1 and galectin-3 were observed in contused brain tissue (M–O). Clotted blood from a neurosurgical (brain) operative field was used to identify peripherally-derived macrophages that strongly stained for both Iba1 and galectin-3 (P–R). Scale bars are 25 μm. Unpublished data.

Figure 4

Coexpression of Iba1 and P2Y12. Human brain biopsy samples from the superior frontal gyrus after TBI were stained with the primary antibodies: goat anti-Iba1 (1:500, Novus Biologicals, Cat No. NB100-1028) and rabbit anti-P2Y12 (1:200, AnaSpec, ANA55043A) followed by goat anti-Alexa Fluor 488 and rabbit anti-Alexa Fluor 594, respectively. Strong coexpression was observed in Iba1+ microglia with P2Y12 in both ramified (A–C) and activated state (D–L) in the non-contused brain tissue. Predominantly amoeboid microglia stained with Iba1 and P2Y12 were observed in contused brain tissue (M–O). Clotted blood from a neurosurgical (brain) operative field was used to identify peripherally-derived macrophages that showed strong Iba1+ but lacked P2Y12+ immunostaining (P–R). Scale bars are 25 μm. Unpublished data.

Figure 5

Coexpression of Iba1 and TMEM119. Human brain biopsy samples from the superior frontal gyrus after TBI were stained with the primary antibodies: goat anti-Iba1 (1:500, Novus Biologicals, Cat No. NB100-1028) and rabbit anti-TMEM119 (1:500, Abcam, Cat No. ab185333) followed by goat anti-Alexa Fluor 488 and rabbit anti-Alexa Fluor 594, respectively. Strong coexpression was observed in Iba1+ microglia with TMEM119 in both ramified (A–C) and activated state (D–L) in the non-contused brain tissue. Predominantly amoeboid microglia stained with Iba1 and TMEM119 were observed in contused brain tissue (M–O). Clotted blood from a neurosurgical (brain) operative field was used to identify peripherally-derived macrophages that showed strong Iba1+ but lacked TMEM119+ immunostaining (P–R). Scale bars are 25 μm. Unpublished data.

Figure 6

Concerns with CD11b (Clone OX42) immunostaining for microglia in human tissue. Human brain biopsy samples from the superior frontal gyrus after TBI were stained with the primary antibodies: mouse anti-rat OX42 (1:100, Bio_Rad, Cat. No. MCA275R) (A–I), or mouse anti-human CD11b (1:400, Novus Biologicals, Cat. No. MAB16991) (J–R); then with rabbit anti-Iba1 (1:1000, Wako, Cat No. 019-19741) (J), or rabbit anti-P2Y12 (1:200, AnaSpec, ANA55043A) (M), or rabbit anti-GFAP (1:1000, Dako, Cat No. Z0334) (P), followed by rabbit anti-Alexa Fluor 488 and mouse anti-Alexa Fluor 594 or tyramide amplification with ExtrAvidin FITC for the mouse anti-human CD11b (K,N,Q). Limited anti-rat OX42 expression was observed in Iba1+ microglia with a ramified state morphology (A–C), but some anti-rat OX42 expression in Iba1+ microglia in the more activated state (D–F) or contused brain tissue (G–I). Anti-human CD11b staining was absent in Iba1 (J–L) and P2Y12 (M–O) immunopositive cells, but coexpressed with the GFAP astrocytic marker (P–R). Scale bars are 25 μm. Unpublished data.