Stefano Fumagalli is a fellow of the Monzino Foundation.

1. Immunofluorescence
The following protocol is performed on coronal brain cryosections obtained from transcardially perfused mice (20 ml of PBS, 0.1 mol/liter, pH 7.4, followed by 50 ml of chilled paraformaldehyde 4% in PBS). After perfusion, brains are carefully removed and transferred to 30% sucrose in PBS at 4 &#176;C overnight for cryoprotection. The brains are then rapidly frozen by immersion in isopentane at - 45 &#176;C for 3 min before being sealed into vials and stored at -70 &#176;C u...

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	<li>Davalos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+mediates+rapid+microglial+response+to+local+brain+injury+in+vivo.">ATP mediates rapid microglial response to local brain injury in vivo.</a> Nat. Neurosci. 8, 752-758 (2005).</li><li>Yenari, M. A., Kauppinen, T. M., Swanson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+in+stroke:+therapeutic+targets.">Microglial activation in stroke: therapeutic targets.</a> Neurotherapeutics. 7, 378-391 (2010).</li><li>Iadecola, C., Anrather, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunology+of+stroke:+from+mechanisms+to+translation.">The immunology of stroke: from mechanisms to translation.</a> Nat. Med. 17, 796-808 (2011).</li><li>Jin, R., Yang, G., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mechanisms+in+ischemic+stroke:+role+of+inflammatory+cells.">Inflammatory mechanisms in ischemic stroke: role of inflammatory cells.</a> J. Leukoc. Biol. 87, 779-789 (2010).</li><li>Schilling, M., Besselmann, M., Muller, M., Strecker, J. K., Ringelstein, E. B., Kiefer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+phagocytic+activity+of+resident+microglia+over+hematogenous+macrophages+following+transient+focal+cerebral+ischemia:+an+investigation+using+green+fluorescent+protein+transgenic+bone+marrow+chimeric+mice.">Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice.</a> Exp. Neurol. 196, 290-297 (2005).</li><li>Perego, C., Fumagalli, S., De Simoni, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+pattern+of+expression+and+colocalization+of+microglia/macrophage+phenotype+markers+following+brain+ischemic+injury+in+mice.">Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice.</a> Journal of Neuroinflammation. 8, 174-193 (2011).</li><li>Zanier, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+umbilical+cord+blood+mesenchymal+stem+cells+protect+mice+brain+after+trauma.">Human umbilical cord blood mesenchymal stem cells protect mice brain after trauma.</a> Crit. CareMed. 39 (11), 2501-2510 (2011).</li><li>Capone, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere+derived+cells+exert+a+neuroprotective+action+by+changing+the+ischemic+microenvironment.">Neurosphere derived cells exert a neuroprotective action by changing the ischemic microenvironment.</a> PLoS ONE. 2, e373 (2007).</li><li>Bhatia, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+host+defense+against+Aspergillus+fumigatus+involves+alveolar+macrophages+with+a+predominance+ofalternatively+activated+phenotype.">Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance ofalternatively activated phenotype.</a> PLoS One. 6, e15943 (2011).</li><li>Raes, G., Noel, W., Beschin, A., Brys, L., de Baetselier, P., Hassanzadeh, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FIZZ1+and+Ym+as+tools+to+discriminate+between+differentially+activated+macrophages.">FIZZ1 and Ym as tools to discriminate between differentially activated macrophages.</a> Dev. Immunol. 9, 151-159 (2002).</li><li>Gesuete, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+C1+inhibitor+in+brain+ischemic+injury.">Recombinant C1 inhibitor in brain ischemic injury.</a> Ann. Neurol. 66, 332-342 (2009).</li><li>Sica, A., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+plasticity+and+polarization:+in+vivo+veritas.">Macrophages plasticity and polarization: in vivo veritas.</a> J. Clin. Invest. 122 (3), 787-795 (2012).</li></ol>

We present here immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate localization and co-expression of M/M phenotype markers into the ischemic area (for a more detailed analysis see ref 6). This method combines specific staining of relevant marker of M/M activation with three-dimensional confocal imaging. The fine tuning of antibodies, serum and fluorconjugated working dilutions allows optimal signal to noise ratio of the investigated m...

After acute brain injury, microglia are rapidly activated and undergo dramatic morphological and phenotypic changes1-3. This intrinsic response is associated to recruitment of blood-born macrophages which migrate into the injured brain parenchyma4,5. The role of microglia and macrophages which are antigenically not distinguishable (henceforth referred to as M/M) in brain injury is still debated. An increasing number of studies indicate that, similarly to what described for peripheral macrophages, microglia and brain recruited macrophages can assume different phenotypes whose extremes correspond to classic pro-inflammatory toxic (M1) or anti-inflammatory protective (M2) phenotype. The different activation states, including secretion of pro- or anti-inflammatory factors, release of neurotrophic molecules and lysosomal activity are characterized by a specific pattern of phenotypic markers, whose expression depends on the temporal evolution of the surrounding environment. The characterization of these M/M phenotypes in the injured brain is still scanty. We used a well-established murine model of pMCAo to analyze M/M expression and evolution after stroke. Immunofluorescence based protocols presented here aim at getting insight into the appearance of specific M/M phenotype markers, their localization and cellular co-expression in the ischemic area. We investigated a few molecules associated to different activation state or phenotype, namely CD11b, a surface marker expressed by leukocytes and a widely used marker of M/M activation/recruitment6-8, CD68 a marker of lysosomes6,7 and Ym1 a secretory protein expressed by alternatively activated (M2) macrophages and associated to recovery and function restoration9-10.	
	When two markers are expressed by the same cell, but in different subcellular compartments, colocalization alone may not be much informative. In this case, analysis of coexpression can be performed by using single plane view and by three-dimensional renderings. We here describe a protocol to obtain a thorough three-dimensional analysis of marker coexpression.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD11b</td>
			<td>Kindly provided by Dr. A. Doni, Istituto Clinico Humanitas, Milan, Italy</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD68 </td>
			<td>AbD Serotec</td>
			<td>MCA 1957</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rabbit Anti-mouse Ym1 </td>
			<td>Stem Cell Technologies</td>
			<td>1404</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hoechst 33342</td>
			<td>Life technologies</td>
			<td>H21492</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse Anti-Neural Nuclei (NeuN)</td>
			<td>CHEMICON</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biotinilated Goat Anti-Rat antibody</td>
			<td>Jackson Immuno Research</td>
			<td>112-065-143</td>
			<td></td>
		 </tr>
		 <tr>
			<td>TSA Cyanine 5 System </td>
			<td>Perkin Elmer</td>
			<td>NEL705A001KT</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Prolong Gold </td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rat alexa 546</td>
			<td>Invitrogen</td>
			<td>A-11081</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse Alexa 488</td>
			<td>Invitrogen</td>
			<td>A-21121</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rabbit Alexa 594</td>
			<td>Invitrogen</td>
			<td>A-11037</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Normal Goat Serum</td>
			<td>Vectors Laboratories</td>
			<td>S-1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tritin X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma</td>
			<td>P4417-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Cryostat CM1850</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Olympus IX81 confocal microscope </td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>AnalySIS software</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaris software 5.0</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Photoshop cs2</td>
			<td>Adobe Systems</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software packages GraphPad Prism version 4.0</td>
			<td>GraphPad Software Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

three-dimensional confocal analysis of microglia/macrophage markers of polarization in experimental brain injury

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.	
	Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 &mu;m z-axis and a 0.23 &mu;m step size, corresponding to a 180 x 135 x 10 &mu;m volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

An example of the results obtained when labeling protocols and confocal acquisitions are carried out into the ischemic region is illustrated in Figures 1A and 1B. A two dimensional view of acquired images shows that at twenty-four hours after ischemia (A), the lysosomal marker CD68 (green) is expressed in hypertrophic ameboid CD11b cells (red) present in the ischemic core. In the border zone (B) CD11b positive cells display round cell bodies and ramified...

Watch this Scientific Journal Video about Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury at JoVE.com

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

three-dimensional-confocal-analysis-microgliamacrophage-markers

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JoVE Journal

Neuroscience

11.5K Views.  IRCCS - Istituto di Ricerche Farmacologiche Mario Negri. A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.