ForDNAanalysis:SpecialLCMprocessingscheduleisfollowed.ThewaterbathiscleanedusingRNAseZap™,rinsedthoroughlywithtripledistilledwater.Thewaterbathisfilledwithtripledistilledwater.Allinstrumentsusedarecleanedwith100%EtOH(brushes,forceps,pencil).Sectionsnotusedaretoweledoffwithkimwipesimmediately.Anewbladeisusedforeachblockand100%EtOHisusedtowipeeachbladewhilesectioning.Glovesarewornatalltimeswhilesectioningandfreshglovesareputoneveryfifthblock.Talkingiskepttoaminimumwhilesectioningtoavoidcontaminationofarea.

ForRNAandProteinanalysis:Cryostatfrozensectioningused.Bladeiswipedwith100%EtOHbetweenblocksanddried.Cryostatareaandallinstrumentsarecleanedwith100%EtOH.Allinstrumentsusedarecleanedwith100%EtOH(brushes,forceps,pencil).Anewbladeisusedforeachblockand100%EtOHisusedtowipeeachbladewhilesectioning.Glovesarewornatalltimeswhilesectioningandfreshglovesareputoneveryfifthblock.Talkingiskepttoaminimumwhilesectioningtoavoidcontaminationofarea.

StainingofParaffinSections

ParaffinembeddedtissueshouldbecutperguidelinesandusedassoonaspossIBLeforbestresults.ThisprotocolhasbeenusedbyseveralonsiteinvestigatorswithgoodresultsforPCR.

1.Xylene(5minutes)2.Xylene(5minutes)3.100%EtOH(30seconds)4.100%EtOH(30seconds)5.95%EtOH(30seconds)6.Distilledwater(15seconds)7.Mayer’sHematoxylin(15seconds)Filteredbeforeeveryuse!8..5%LithiumCarbonateindistilledwater(30seconds)9.Distilledwater(30seconds)10.70%EtOH(30seconds)11.Eosin(30seconds)Filteredbeforeeveryuse!(alcoholicEosinYwithPhloxine)12.95%EtOH(15seconds)13.100%EtOH(30seconds)14.100%EtOH(30seconds)15.Xylene(1minute)16.Xylene(1minute)Airdrycompletely(2-4minutes)

NIHprotocolmodifiedbyNIEHS

StainingofFrozenSections

Frozentissueshouldbecutperguidelinesandstainedassoonaspossibleforbestresults.ThisprotocolhasbeenusedbyseveralonsiteinvestigatorsforRNA,DNAandProteinanalysis.*70%or95%EtOHisthepreferredfixativeforLCMsamples.1minuteforfixationthenstartslidesatstep#6perstainingprotocol.

6.Distilledwater(15seconds)7.Mayer’sHematoxylin(15seconds)Filteredbeforeeveryuse!8.1xAutomationBuffer(30seconds)9.Distilledwater(30seconds)10.70%EtOH(30seconds)11.Eosin(30seconds)Filteredbeforeeveryuse!(alcoholicEosinYwithPhloxine)12.95%EtOH(15seconds)13.100%EtOH(30seconds)14.100%EtOH(30seconds)15.Xylene(1minute)16.Xylene(1minute)Airdrycompletely(2-4minutes)

Mayer’sHematoxylinCat#MHS16-500mlSigmaEosinYCat#1B425CellPointScientific25gramPhloxiNEBCat#P-387Daigger(Fisher)1xAutomationBuffer(Fisher)

NIHprotocolmodifiedbyNIEHS

ThumbnailImages

ProcedureForPrintingThumbnails/ImagesFromNIEHSLCM

1)StartARC-100softwareprogramandadvancetoLCMSystemUserSelection2)Highlightbyclickingonthestudynamedesired3)PressReviewData4)Chooseappropriatefolder(s)5)PressThumbnails6)Scrolltoputdesiredimage(s)inview(ifasingleimageisdesiredyoucanopenitbydoubleclickingontheimageandproceding)7)DoubleclicktheShortcuttoSnap32iconondesktop.8)ClickOK9)PressCtrl+F11keys.Ablinkingframewillappeararoundthethumbnailstobeprinted.10)Clickonblinkingpicture11)Hypersnapfinderwithnewthumbnailpicturewillopen12)UnderFile(menu)choosePrintSetuptosetparameters.13)Controlkey+Ptoprintimages14)CloseandExitallwindowsplease.