Page - 20 - in Advances in Neuroimmunology

BrainSci. 2016,6, 15 2.3. IHCandHCAnalysis Embeddedparaffintissueswerecut into8μm-widesections inamicrotome(Leica,Germany) andmounted inSuperfrost slides (ThermoScientific,Waltham,MA,USA). Microgliawere identifiedbylectinHC(tomato lectin fromLycopersiconesculentum, Sigma-Aldrich 1:500 inTris-bufferedsaline (TBS)with0.5%tritonX-100 (TBS-t)).Astrocyteswere identifiedbyGFAP IHC(rabbitanti-GlialFibrillaryAcidicProtein fromDakoCytomationDenmark,1:1200 inblocking buffer). Lymphocyteswere identifiedbyCD3 IHC(rabbit anti-humanCD3,DakoA0452, 1:100 in blockingbuffer). Spinalcordsectionswerealsostainedwithhematoxylin-eosinandwithLuxolFast Bluesolution(LFB) (0.1%,overnightal37˝C)andcounterstainedwithCresylviolet (0.1%,1min), to assess thenumberofcellular infiltratesanddemyelination ingreymatter, respectively. Sections for IHC andHCwere preincubated for 1 hwith the blocking buffer (0.5%BSA in TBS-t) and then incubatedwith theprimaryantibodies (GFAP,CD3)or tomato lectinovernight at 4 ˝C, followed by 1 h at room temperature (RT) (GFAP,CD3) or at 37 ˝C (lectin). ForCD3 IHC, apreviousantigenretrieval stepwasperformedwithprotease typeXIV(SigmaP5147).Afterwashing inTBS, sectionswere incubatedwitheitherhorseradishperoxidase-coupledstreptavidin (VectorLabs, Burlingame,CA,USA,1:600,1h) forHCorbiotinylatedsecondaryantibody(VectorLabs,Burlingame, CA,USA, 1:300, 1 h atRT) followedbywashes andhorseradishperoxidase-coupled streptavidin (VectorLabs, Burlingame,CA,USA, 1:600, 1h) for IHC.The immunoreactivitywasvisualizedby using 0.033%H2O2 in 0.5mg/mL3,3-diaminobenzidine-tetrahydrochloride (DAB) in Tris buffer (TB) for 4–30minat room temperature. Reactionwas stoppedwithTB,washed, dehydrated and mounted inDPX(distyrene-plasticiser-xylene) (Sigma,StLouis,MO,USA). Imagesat100ˆ (GFAP)or 200ˆ (lectin, CD3)were taken in a bright fieldNikonEclipse 90imicroscope (Nikon Instruments EuropeBV,Amsterdam, TheNetherlands) and acquiredwith aNikondigital cameraDXm1200F (NikonInstrumentsEuropeBV,Amsterdam,TheNetherlands)andNikonAct-1version2.70software (NikonInstrumentsEuropeBV,Amsterdam,TheNetherlands) fromdifferentbrainareasandspinal cord. Finally, to quantify staining areas, intensity and thenumber of cells, imageswere analyzed usingImageJsoftware (NIH,Bethesda,MD,USA).Histologicalanalyseswereperformedonat least twosectionspermouse.Control sections fornon-specificbindinganalysis (whereprimaryantibodyor tomato lectinwasnotused)were includedroutinely. ForGFAPIHC, thepercentageofstainedarea (at100ˆ)wasmeasured indifferent regionsof the spinal cordfromEAE-inducedmice. Six to twelve imagesperanimalwereexamined.Whiteandgrey matter areasof spinal cordswerealsoanalyzed (12 imagesperanimalandarea). A thresholdwas set foreacharea tobetterdefinecells fromtissuebackground.Because tomato lectinHCstainsboth microgliaandvessels, thequantificationof stainingusing ImageJwassupplementedwithmanual countsofmicroglial cells showingabasal (ramified), reactiveor fully-activated(round)morphology andof thenumber of vessels; thesewere assessed in the same regions as forGFAP IHCanalysis, at 200ˆ. Inaddition, totalnumbersofmicroglia/macrophage infiltrationareas inspinal cordwere recorded. ForCD3IHC, thepercentageofstainedareawasmeasured in thespinal cord(10 imagesper areaandanimal). InLFB/Cresylviolet staining, totalnumbersofcellular infiltrates ingreymatterof spinalcordwerecounted,andafterwards,acolordeconvolutionplugin fromImageJsoftwarewas usedinorder toseparatecolors fromLFBandCresylviolet tobeable toquantify thedemyelinationby analyzingthepercentageofLFB-stainedarea inspinalcord. Thethresholdsetgave100%ofareaof the spinalcordcoveredbyLFBstainingforhealthyfemalemice. Tostandardize, thesamethresholdwas usedfor femaleandmalemice.Demyelinationandadecreaseof theareacoveredbyLFBcausedby EAEwerereadilydetected inbothmaleandfemalemice. 2.4. StatisticalAnalysis Values in text and figures are shown as the mean ˘ standard error of the mean (SEM). StatisticalanalyseswereperformedusingSPSSstatistical software (version17.0 forWindows,SPSS Inc.,Chicago, IL,USA).pď0.05wasconsideredsignificant inallanalyses. 20