Laser capture microdissection

From VLP

Cryosectioning

 * Clean cryostat with EtOH to make RNase-free
 * Use PEN-foil membrane slides (Leica #11505158, order from JT Technologies)
 * Cut at 7-10µm thickness (7µm best)
 * Can place 7-10 sections per slide
 * Need ~8 slides per sample (depending on what you’re isolating)
 * Keep slides on dry ice or in cryostat AT ALL TIMES
 * Store slides at -80ºC—keep as dry as possible
 * Slides should be used within 4 days of sectioning

Staining :

 * Use LCM Staining Kit (Ambion #AM1935)
 * Follow protocol that comes with kit with minor modifications (work quickly to rehydrate/dehydrate tissue)
 * Setup: Fill chambers with ~30ml EtOH and place in 50ml Falcon tube rack; make paper towel blotter; get Cresyl violet out of 4ºC walk-in
 * Briefly:
 * Take slides out of -80ºC and place on dry ice
 * Quickly place in 95% EtOH 30-40s while moving slides up and down with forceps and remove excess EtOH by tapping on kimwipe
 * Transfer slides to 75%EtOH chamber for 30-40s while moving slides up and down and remove excess EtOH by tapping on kimwipe
 * Transfer slides to 50%EtOH chamber for 25-30s while moving slides up and down and remove excess EtOH by tapping on kimwipe
 * Mark area to be stained with barrier pen making sure not to touch foil area
 * Cover area with 300-800µl cresyl violet stain for up to 1.5min
 * Dump stain on paper towel blot and tap on kimwipe
 * Place stained slide in 50%EtOH chamber for 30-40s and move up and down several times, remove excess on kimwipe
 * Transfer slides to 75%EtOH chamber for 30-40s and move up and down several times, remove excess on kimwipe
 * Transfer slides to 95%EtOH chamber for 30-40s and move up and down several times, remove excess on kimwipe
 * Transfer slide to 100%EtOH for 30-40s, remove excess EtOH on kimwipe
 * Transfer slides to 2nd 100%EtOH chamber for up to 2 min, remove excess EtOH on kimwipe
 * Transfer slide to xylene moving up and down to remove EtOH
 * Transfer slide to 2nd xylene chamber and incubate for 5 min
 * Remove slides from xylene and air-dry in hood against box for 10 min

LCM :

 * Turn on microscope in ordered labeled (controller 1st, laser 2nd)
 * Turn on computer and open “Leica Microdissection 4.4” software
 * Make sure tube and slide holder are not in
 * Clean tube and slide holder with RNase-zap
 * Place slide upside-down in slide holder
 * Click “unload” iconàplace slideholder in and click into place
 * Fill tube holder with PCR tubes and push into place under slide (snaps into place)
 * Click “continue” on open window
 * Use the controller to change magnification, focus, move platform, adjust lighting (Only use the controller to modify these settings)


 * Calibrate the laser in the objective used for cutting: Click on the “Laser” drop-down menuà “calibrate” (make sure laser is not near tissue)àline up arrows for calibration
 * Bring PCR tube used for collection by selecting icon at bottom of screen—cap should go from red to green and color of selection line will match cap letter color
 * Click on “line” iconàselect “closed lines” and “combined mode”à “start cut”
 * If section cut does not fall into cap, use the “move and cut” function to manually direct the laser—may need to refocus to properly cut


 * To check if cut section fell into cap, click on “collector” icon to inspect cap—should see small pieces of tissue (Do not change objective in this mode)


 * To change intensity, speed or thickness of laser, click on “Laser” drop-down menu and “control”
 * Click on “unload” icon to change slides or PCR tubes
 * CAREFULLY remove PCR tube from tube holder (can add lysis buffer to cap prior to removal)
 * Place tissue on dry ice and store at -80ºC or immediately extract RNA
 * For human epidermis, need to collect 500K-1000K µm2 for ~50-100ng RNA

RNA Isolation :

 * Use RNAqueous-Micro kit (Ambion #AM1931)
 * Follow kit protocol, Section B: RNA isolation protocol from LCM sampes
 * Modifications:
 * Step 4: add 1.25 volumes 100% EtOH
 * Step 9: elute with 2x8µl elution solution
 * Store at -80ºC

RNA Amplification :
Use 10µl RNA per reaction (should get ~10-12µl from RNA isolation step—generally use all RNA for amplification—may need to do 2 rounds)

Include DNase treatment after in vitro transcription if downstream application is qRT-PCR