Table 2.
The commonly respective used fluorescent labeling methods for vessels, astrocytes and microglial cells
| Visualization strategy | Visualization methods | Comments | Reference | |
|---|---|---|---|---|
| Vessels | ||||
| Structure labeling | Intravenous injection fluorescently labeled vascular tracers, e.g., Evans blue, Fluorophore conjugated dextran (including FITC-dextran, Texas red-dextran, and Alexa 680-dextran) | Fluorescent label the serum | Dirnagl et al., 1992; Rovainen et al., 1993; Kubotera et al., 2019 | |
| Intravenous injection plant lectin conjugated with fluorophores, e.g., tomato lectin, FITC-wheat germ agglutinin lectin | Imaging the endothelial glycocalyx. | Robertson et al., 2015; Yoon et al., 2017 | ||
| Tg eGFP-Claudin5 transgenic mice | Imaging the tight junction between endothelial cells. Expressing eGFP and Claudin5 under the direction of the endothelial-specific receptor tyrosine kinase (Tek, formerly, and Tie2) promoter. |
Knowland et al., 2014 | ||
| Tie2-GFP transgenic mice or crossing Tie2-Cre with floxed fluorescent protein reporter mouse line | Expressing GFP under the direction of the endothelial-specific receptor tyrosine kinase (Tek, formerly, Tie2) promoter. Widely used in imaging the endothelial cells. The homozygous Tie2-GFP mice have retinal degeneration and are blind in adulthood, which may affect their performance in behavioral tests after ischemic stroke. |
Motoike et al., 2000; Kisanuki et al., 2001 | ||
| Crossing Tie2-CreER transgenic mice with floxed fluorescent protein reporter mouse line | Forde et al., 2002 | |||
| NG2DsRedBAC transgenic mice or crossing NG2-CreERTM with floxed fluorescent protein reporter mouse line. | Expressing fluorescent protein driven by the promoter for NG2 proteoglycan. Mice express red fluorescent protein, DsRed, driven by the control of promoter for NG2 proteoglycan. Labeling mural cells as well as polydendrocytes and oligodendrocyte progenitor cells. |
Hartmann et al., 2015b | ||
| PDGFRβ-tdTomato or PDGFRβ-CreERT2 transgenic mice | Expressing fluorescent protein driven by the promoter for PDGFRβ, labeling mural cells. Cannot separate pericytes and smooth muscle cell. |
Hartmann et al., 2015b | ||
| αSMA-mChrerry or crossing αSMA-CreERT2 with floxed fluorescent protein reporter mouse line | Expressing fluorescent protein driven by the promoter for αSMA, labeling mural cells. | Wendling et al., 2009; Armstrong et al., 2010 | ||
| Functional labeling | Intravenous injection fluorescent vascular tracers, including: Fluorophore conjugated dextran, e.g., FITC-dextran Texas red-dextran Alexa 680-dextran | Fluorescent label the serum. The velocity and direction of blood flow are calculated from the unlabeled red blood cell flow paths through time lapse 2PLSM imaging. |
Ngai and Winn, 1996; Kleinfeld et al., 1998; Saunders et al., 2015 | |
| Crossing PDGFRβ-CreERT2 with Rosa26< LSL-GCaMP6s> (Ai96) transgenic mice | Expressing GCaMP6s driven by the promoter of PDGFRβ. GCaMP cpGFP, CaM, and the Ca2+/CaM-binding M13pep. Intracellular calcium concentration fluctuations shown by the fluorescence intensity. Imaging the fluctuation of intracellular calcium in mural cells. |
Glück et al., 2021 | ||
| Acta2-RCaMP1.07 | Expressing RCaMP1.07 driven by the promoter of αSMA. RCaMP1.07 is a red fluorescent protein with a calmodulin binding domain. |
Meza-Resillas et al., 2021 | ||
| Intravenous injection of fluorescently labeled vascular tracer that does not cross the blood-brain barrier under normal conditions, e.g., Evans blue, fluorophore conjugated dextran (including FITC-dextran, Texas red-dextran, Alexa 680-dextran), Alexa-albumin | Calculation the permeability of BBB. | Saunders et al., 2015; Ahishali and Kaya, 2021 | ||
| Astrocytes | ||||
| Structure labeling | Direct application of dyes to the cortical surface or intravenous injection sulforhodamine 101 (SR101) sulforhodamine B (SRB) | Not very specific for labeling astrocytes. | Nimmerjahn et al., 2004; Vérant et al., 2008a; Appaix et al., 2012; Hill and Grutzendler, 2014 | |
| GFAP-GFP or GFAP-eGFP transgenic mice | Expressing GFP or eGFP driven by the promoter of GFAP. Only reactive astrocytes are labeled. Simultaneous labeling of neurons. |
Nolte et al., 2001 | ||
| Aldhlll-eGFP | Expressing eGFP dricen by the aldehyde dehydrogenase 1 family member L1 (Aldh1l1) promoter/enhancer regions. | Mills et al., 2022 | ||
| Functional labeling | Direct coloading Fluo-4 acetoxymethyl (AM) ester to the cerebral cortex | Imaging in vivo cytosolic calcium fluctuation in astrocytes. | Hirase et al., 2004 | |
| Injection the Ca2+-sensitive fluorescent indicator Oregon Green 488 BAPTA-1 (OGB-1) and SR101 into the brain. | Not very specific for labeling astrocytes. | Ma et al., 2021 | ||
| Crossing Aldh1l1-Cre/ERT2 transgenic mice with floxed fluorescent protein reporter mouse line or stereotactic injection Cre-dependent FLEX-GFP AAVs in Aldh1l1-Cre/ERT2 BAC transgenic mice | Srinivasan et al., 2016 | |||
| Microglial cells | ||||
| Structure labeling | Incubating brain slices in FITC-IB4 | Imaging microglia in living brain slices and slice cultures | Dailey et al., 2013 | |
| Intravenous injection plant lectin conjugated with fluorophores (tomato lectin, isolectin IB4) | No need to breed transgenic mice. Quick and easy marking. Labeled volumes are limited. Simultaneous labeling of partial endothelial cells. Longitudinal imaging is not possible since the requirement for intraparenchymal injection of the fluorophore. |
Brawek and Garaschuk, 2017 | ||
| Intravenous injection peridinin chlorophyll conjugated Iba-1 and aminomethyl coumarin acetate conjugated CD68 into wild type mice before 2PLSM | No transgenic mice required. Blocking receptors on microglia. |
Bok et al., 2015 | ||
| F4/80 or CD68 promoter in combination with the surface-mutated AAV6 capsid | Selective GFP expression in vivo after intracerebroventricular injections in wild-type mice. May induce pathological conditions. | Rosario et al., 2016 | ||
| LV.miR-9.T, microRNA-9-regulated vector | Microglia of rodent brain do not express miR-9. Exclusion of peripheral bone marrow cells. Not entirely selective, depending on cell expression level of microRNA-9. |
Åkerblom et al., 2013 | ||
| CX3CR1-GFP transgenic mice CX3CR1-EGFP transgenic mice | Expressing GFP or EGFP sequence replacing the first 390 bp of the coding exon (exon 2) of the chemokine (C-X3-C motif) receptor 1 (Cx3cr1) gene. The most widely used fluorescent labeling techniques of microglia. Simultaneous labeling microglia and peripheral monocytic cells. Co-imaging microglia and neurons requires cross with Thy1-GFP transgenic mice, thus neurons and microglia have the same fluorophore (GFP) and may not be well distinguished from neurons by morphology. Partial knockdown of CX3CL1/fractalkine pathway, which may influence the pathophysiological process. |
Jung et al., 2000; Davalos et al., 2005; Nimmerjahn et al., 2005; Wake et al., 2009; Goldmann et al., 2016; Wieghofer and Prinz, 2016; Haimon et al., 2018 Fumagalli et al., 2013; Brawek and Garaschuk, 2017; Hickman et al., 2019 | ||
| Iba1-EGFP transgenic mice | Expressing the EGFP under the direction of Iba1 gene. Widely used fluorescent labeling technology of microglia. Simultaneous labeling microglia and peripheral monocytic cells. Weak GFP fluorescence expression. Iba-1 expression is unstable in the same cells, correlating with Iba-1 protein expression level. |
Hirasawa et al., 2005 | ||
| CD11b-CreERT2 cross R26-tdTomato transgenic mice | Only labels partial microglia. Simultaneous labeling of microglia and peripheral blood mononuclear macrophages. |
Füger et al., 2017 | ||
| CD11b-CreERT2;R26-tdTomato;Iba1-eGFP | Labels all microglia. Simultaneous labeling of microglia and peripheral blood mononuclear macrophages. |
Füger et al., 2017 | ||
| Tmem119-tdTomato reporter mouse Tmem119-EGFP and Tmem119-CreERT2 transgenic mice | Expressing tdTomato or EGFP driven by the promoter of TMEM119. Specifically label microglia. Not affect cell function and physiological function. Using the CRISPR-Cas9 technology. |
Ruan et al., 2020 Kaiser and Feng, 2019 | ||
| Functional labeling | Staining calcium in microglia with OGB-1 using a single-cell electroporation technique | First fluorescent labeling of intracellular calcium signals in microglia. Failing to render calcium signaling changes in the entire microglial cell network. |
Eichhoff et al., 2011 | |
| Injection of recombinant retroviral vector to introduce GCaMP2 | Labeling microglia network calcium signaling. Retroviruses infect only dividing cells; stab wound injury is required to stimulate microglia proliferation. | Seifert et al., 2011 | ||
| Twitch-2B in microglia by a microglia-specific microRNA-9-regulated viral vector | Expression of a genetically-encoded ratiometric Ca2+ sensor Twitch-2B in microglia. Not entirely selective, depending on cell expression level of microRNA-9. |
Brawek et al., 2017 | ||
| A Cre-dependent GCaMP5G line, termed PC::G5-tdT (Polr2a, CAG, GCaMP5G, tdTomato) | No effect on physiological functions of transgenic mice. Visualizing calcium signaling of microglia. Low expression level of calcium fluorescence signal. Need to breed transgenic animals. |
Gee et al., 2014 |
2PLSM: Two-photon fluorescence laser-scanning microscopy; AAV: adeno-associated virus; BBB: blood-brain barrier; CaM: calmodulin; cpGFP: circularly permuted green fluorescent protein; eGFP: enhanced green fluorescent protein; FITC: fluorescein isothiocyantate; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; Iba1: ionized calcium-binding adaptor molecule 1; M13pep: “M13” peptide; NG2: neural/glial antigen 2; PDGFRβ: platelet-derived growth factor receptor β; TMEM119: transmembrane protein 119; αSMA: α-smooth muscle actin.