SlP ICid ISample Preparation Considerations For Laser MicroDissection

SSamplel PreparationPi ConsiderationsC id ifor Laser MicroDissectionAll contents Copyright 2012 by Leica Microsystems,Inc.ABRF Meeting, March 2012

What are the Considerationsfor Sample Preparation for LCM / LMD?Staining:Tissue:ssue Tissue type Thickness Target cellsLCM / LMDSystem: Lasercapabilities?p Laser settings? Protocols? How toidentifytarget cells?Slide: Whattype? treatmentDownstream: Analysis? Kits? Technique?LiveCell?

Leica LMD Protocol Guide

Staining Protocols Less is More Adjust protocols for minimal stain to allow identificationof target cells. Less stain can improvepcontrast in the image.g Use no stain. Use stained sections on one slide as a navigational toolt identifytoid tif areas off interesti tt on unstainedt i d sectionstionanother slide Use software module to transfer the regions of interestfrom a properly stained section on one slide tounstained serial sections on other slides. Fluorescent tags require special consideration.

Typical Sample Preparationfor Laser Microdissection forFrozen Sections for Animal Cells Cut sections 4-20 umMount on chosen slidesFixation with 70% EtOH for 30 sec. minimumWash in DEPC water for 30 sec.Stain withith MaMayer’ser’s hematoxlinhemato lin for 30 secsec.Wash in DEPC water for 30 sec.Counterstain with eosin for 30 secsec.Rinse with (95%) and (100%) EtOH for 30 sec.Air dryy approximatelyppy 5 minutes

FixationPreserve the tissues; preserve the macromolecules; stop metabolism;allow visualization identification; better LMD.Recommended Cryofixation, a goodchoice Fixation by ice-coldacetone, 30 – 45seconds Fixation by gradedalcohols up to 70%Other Xylene fixation forshort times. (Xylenesubstituteb tit t nottrecommended.) Formalin fixation @4% will cross link RNA, Good fixation will allowfor better LCM/LMD

FlFluorescenceConsiderationsC id ti OOver-dehydrationd h d ti bby AlAlcohols,h l or XXylenelmay cause bboththimmunofluorescence and fluorescent proteins (e.g. GFP)to deactivate. Recommend series of alcohol fixation/dehydration up to70% Ethanol. Recommend short time fixation/dehydration in Xylene. GFP is a common fluorescent protein tag; it varies widelyby manufacturer.manufacturer Contact with polymers may cause GFPto deactivate. Use a coating (e.g. poly-l-lysine) as aprotectant. Choose a low auto-fluorescent slide (e.g. PPS, FLC, EP).

Choose the Right Slide Systems using foil slides: PET (polyethylene terephthalate) recommended forMALDI, SELDI ionization analysis. PEN ((polyethylenel th lnaphthalate)hth l t ) nott recommendedd d ffor anyionization analysis. Expression Pathology EP Director slides recommendedfor proteomic analysis, for large areas; low autofluorescence background. LowLflfluorescence bbackgroundkd slideslid withith PPSPPS, FLCFLC, EPEP.

PPre-treatingi SlidesSlid UV irradiation prior to mounting specimen has beneficialeffects:- Makes all slides RNase, DNase, Protease free andsterile.- MakesM k ffoilil slideslid more adherentdht ffor titissue.- Makes laser cutting of foil slides more efficient.

Know your LCM / LMD System What are the capabilities and limitations of the laser forcutting ROI’s? WhatWh t iis ththe maximumisamplel thithicknesskyour systemtisicapable of cutting/capturing? How can you optimize the system parameters forcutting/capturing your particular specimen? How can you save the laser parameters?

SSomeRReferencesfCitations:Proteomic Analysis of Cells Isolated by Laser Microdissection, V. Fiorilli, V. Klink, et.al., Chapter 3, “Integrative Proteomics”, Intech, 2012, pp 47 – 69.Desmin expression in colorectal cancer stroma correlates with advanced stagedisease and marks angiogenic microvessels, G. Arentz, et. al., Clinical Proteomics2011, 8:16Increased Phospho-KeratinPhospho Keratin 8 Isoforms in Colorectal TumorsAssociated with EGFR Pathway Activation and Reduced Apoptosis, G. Arentz, et. al.,Molecular Biology Volume 2012, Article ID 706545MALDI-TOFMALDITOF and MALDIMALDI-FTICRFTICR imaging mass spectrometryspectrometr of methamphetamineincorporated into hair, AkihiroMiki, et. al., J. Mass. Spectrom. 2011, 46, 411–416.Medullary amyloidosis associated with apolipoprotein A-IV deposition, S. Sethi, et. al.,Kidney International 2011 International Society of Nephrology,

R 7000/http://www.expressionpathology.com/pdf/iTRAQ labeling of Liquid Tissue Preps uidTissueManualV146.pdf/// rature.aspx?id tml

Desmin expression in colorectal cancerexpression in colorectal cancer stroma correlates with advanced stagecorrelates with advanced stage disease and marks angiogenic microvessels, G. Arentz, et. al., Clinical Proteomics 2011, 8:16 Increased Phospho-Keratin 8 Isoforms in Colorectal TumorsKeratin 8 Isoforms in Colorectal Tumors