1

Laser Microdissection Analysis Platform

Administrative Coordination:M. Nakache (CR1)

Scientific Coordination: T. Jay (DR2) & L. Villanueva (DR2)

Experimental Coordination: E. Davenas (IR1)

2

Laser Microdissection : a tool within a process

Extraction

(2)

HistologicObservations

Histochemical-immunity In Situ Hybridation TUNEL

(4)

Laser Microdissection

GenomeTranscriptome

PCRRT-PCR

ProteomeWestern blot

2D-gel, 2D-DIGEMass Spectrometry

TissuePrelevement

(1)

(3)localisation

3

Goals

Cell or tissu isolation under morphologic control in order to perform molecular biology studies

from either frozen or paraffin-embedded tissue slices or cell cultures

4

From tissue to molecule (1)

1. Sample preparation and cutting - fixation - paraffin embedding - freezing

2. Visualisation of areas of interest - staining - fluorescence

3. Selective Microdissection - no contact, no contamination - any size (µm2 -> mm2), shape, thickness (< 150 µm)

5

LMPC PALM® System Laser Microdissection & Pressure Catapulting

The laser beam cuts the area of interest then, by adjusting its energy and its focusing, it catapults the selected region into the cap of a tube placed just over. This cap is filled with a lysis buffer adequate for subsequent analysis:

- DNA - RNA - Protein

6

From tissue to molecule (2)

4. Extraction and preparation of specific molecules: DNA, RNA, proteins

5. Analysis of pertinent, reproducible and specific results

7

DNA Extraction Tumor Analysis DNA Fingerprinting

DNA

RNA RNA Extraction Quantitative RT-PCR Microarray analysis

Protein HPLC SDS-PAGE Mass Spectroscopy analysis (MALDI, SELDI)

Molecular analysis: DNA, RNA, Proteins

8

Materials available

PALM® Microbeam Laser Microdissector (Zeiss)

Cryostat HM550 (MM France)

Microtome HM450 (MM France)

UV Crosslinker BLX-254 (BIO-LINK)

Hood Chemfree 90S (Oxygen)

9

Laser Microdissection

2,5X

10X

2,5X

10X

2,5X

10X

Globus Pallidus Subthalamic Nucleus Black Matter