Page - 67 - in Advances in Neuroimmunology

BrainSci. 2016,6, 16 oneof the followingprimaryantibodiesat4˝C:mouseanti-OX-42 (1:1000;SerotecMCA275;Raleigh, NC,USA),mouseanti-ED-1(1:500;SerotecMCA341),mouseanti-OX-6(1:500;Serotec,MC2687),or rabbit anti-Iba-1 (1:1000;Wako, 019-19741; Richmond,VA,USA). Primarieswere chosen for their specificity formicrogliaphenotypes [26,43].OX-42wasselectedasamarkerofmicroglialactivation because it recognizesclusterofdifferentiationmolecule11b/c(CD11b/c)ofcomplementreceptor3 (CR3),which isconstitutivelyexpressed inmicroglia;however,upregulationofCD11b/c isoneof the first indicationsofmicroglialactivation[44–46]. BothED-1andOX-6areselective formoreclassical formsofmicroglial activation [26]. ED-1 recognizes the lysosomalmembranesofmicroglia and is thought tobe an indicationofphagocytic activity [47]. OX-6, however, is anantibodyagainst the majorhistocompatibility complex-II that elicitsT-helper cell activation [26,48]. The Iba-1 antibody wasselectedbecause it recognizesacalciumbindingproteinexpressed inallmicroglia [49]. Sections were incubated insecondaryantibody(biotinylatedhorseanti-mouse, ratadsorbed,orbiotinylated goat anti-rabbit, Vector Laboratories, Burlingame, CA, USA), avidin-biotin-peroxidase complex (ABCElite Kit, Vector Laboratories) and the chromagen, nickel-enhanced 3,31-diaminobenzidine tetrahydrochloride (Polysciences;Warrington,PA,USA),aspreviouslydescribed[22,31]. Following thefinalwash,allprocessedsectionsweremountedontoglassslides,driedandcoverslippedwith Cytoseal® (StephensScientific,Wayne,NJ,USA). During quantification, slideswere coded to ensure the experimenterwas blind to treatment condition. TodetermineOX-42 immunoreactivity, imagesof thehippocampus (Bregma´2.50and ´4.00mm)orentorhinal cortex (Bregma´3.00and´6.00mm)wereobtainedwitha10ˆobjectiveon anOlympusBX-51microscope(Olympus,CenterValley,PA,USA) linkedtoamotorizedstage (Prior, Rockland,MA,USA),microcatorandDP70digital camera(Olympus) [50].OX-42 immunoreactivity wasdeterminedbyopticaldensitywithVisiomorph™(Visiopharm,Hørsholm,Denmark). Subregions of thehippocampus(dentategyrus (DG), cornuamonis (CA1andCA2/3))andtheentorhinalcortex were tracedseparatelyandthepercentareaofOX-42 immunopositivepixelswithineachregionof interestwasdetermined. Immunoreactivitywas thennormalizedto thead libitumcontrolgroupand expressedaspercentofcontrol. ForED-1orOX-6immunohistochemistry,sectionswerequalitativelyassessedinthehippocampus andentorhinal cortexas inpast reports [22]. Todetermine the impactofethanolonmicroglianumber, Iba-1+ cellswere countedwithin thehippocampus and the entorhinal cortex. Iba-1+ cellswithin thesubregionsof thehippocampuswereestimatedbyunbiasedstereologicalmethodsaspreviously reported[22,51].NewCAST™Stereologysoftware (Visiopharmversion3.6.4.0) coupledto thesame OlympusBX-51microscopesystemaboveapplieda70μmˆ70μmcountingframeandcellswere randomlysampledusinga20μmdissectorheightwith2μmguardzoneswithin theCA1(400μmx,y step length),CA2/3(250μmx,ystep length), andDG(250μmx,ystep length). Total Iba-1+cellswere calculatedusingtheequation(1): N “ ÿ Qˆ1{asfˆ1{tsfˆ1{ssf (1) whereQis thenumberofcellscounted,asf is theareasamplingfraction, tsf is the thicknesssampling fraction,andssf is thesectionsamplingfraction[52].Coefficientsoferrorrangedfrom0.011to0.039 andaveraged0.023˘0.001. Fortheentorhinalcortex,microglianumberwasdeterminedusingaprofile countingmethod[53]. Imagesof theentorhinalcortexwerecollectedwitha10ˆobjectiveusingaSPOT Advanced™camera (SPOTImagingSolutions,SterlingHeights,MI,USA). Iba-1+cellswerequantified incollectedimagesbyanautomatedcountingsystem(ImageProPlus6.3;MediaCybernetics,Rockville, MD,USA)andexpressedasmeanIba-1+cells/section˘SEMaspreviouslydescribed[22]. 2.5. EnzymeLinked ImmunosorbentAssay Hippocampusandentorhinal cortex from the right hemispherewereprocessed forELISAas reportedpreviously [22,54]. Briefly, tissueswerehomogenized inan ice-cold lysis buffer (1mLof 67