Laser capture m icrodissection w as carried out on form alin fixed paraffin em bedded (FFPE) material using the PixC ell® !! LCM System (A rcturus Engineering, Inc., CA, USA). Briefly, 4- 7|j.m FFPE histological sections w ere cut and stained w ith a standard hem atoxylin and eosin (H&E) stain. U pon drying they are placed on the PixCell® !! m icroscopic stage and the follow ing protocol is observed (Figure 2.1):
• Enable the laser via the keysw itch located on front o f controller.
Rem ove the CapSure® cassette m odule from the PixCell® !! platform . It should slide out sm oothly over the detents.
Press down on the flanges o f the cassette m odule. Press the end locking pins in to hold the plate down in the load position.
• Slide a CapSure® cartridge, w hich holds four film carriers, into the cassette m odule until it hits a stop. The cassette loads from one end only.
I f m ore than four caps are required, load a second cartridge in the same manner, making sure there is no space betw een the tw o loaded cartridges.
Press down on the flanges o f the loaded cassette. Retract the locking pins and gently raise the cassette to its loaded position.
• The cartridge is now ready to be loaded into the PixCell®II. • Slide the loaded CapSure® cassette m odule to any detent position.
A fter LCM , w hen the CapSure® cartridges are empty, press dow n on the plate and lock into position by pushing the locking pins in. Carefully slide out the spent cartridges and discard.
M ove the joystick to the vertical position. This centres the translation stage, on the optical axis and ensures proper registration o f the sam ple slide relative to the capture area. This also m axim ises the transfer area available for m icrodissection.
• Turn o ff the vacuum chuck via the switch located on the front o f the controller.
Place the slide on the translation stage, covering the vacuum chuckhole. M anually position it to locate the transfer area in the centre o f the field o f view.
Choose the appropriate objective for the desired m agnification. Turn on the vacuum chuck.
Chapter 2 M aterials and M ethods
• A djust illum ination control. • A djust focus control.
Rotate the CapSure® placem ent arm over the tissue sample.
Insert the visualiser by pressing the silver plunger button located above the joystick. A djust the m icroscope light source to obtain a good image on the monitor.
• To perform a m icrodissection, retract the visualiser by pressing the tab located below the plunger button, and re-adjust the m icroscope light source.
• Slide the CapSure® cassette m odule to a detent position, making sure there is a CapSure® cap at the load line.
Rotate the placem ent arm to the cap pick-up position. The arm will autom atically line up with the cap.
• Raise the placem ent arm vertically to rem ove the cap from the cassette module.
• Rotate the placem ent arm to transfer position over the slide. Release the placem ent arm. The cap will autom atically low er onto the slide. U pon contact with the slide, the placem ent arm w ill seat the cap properly on the sample.
W hen the laser is enabled, an aim ing beam is visible on the monitor.
Select the laser spot size w ith the ‘Spot Size A djust’ located on the left o f the laser tower. U se the joy stick or the X Y controls to aim the laser beam on the capture region.
Use the front panel digital controls on the controller to adjust laser param eters. Typical values are:
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<7.5 |am 40m W 450)is ~15 |am 25m W 1.5ms - 3 0 |j.m 20m W 5 ms. Fire the laser by pressing the rem ote thum b switch or the ‘fire’ button on the front o f the controller.
There is an audible beep w hen the laser fires. The activated portion o f the transfer film will be visible as a ring o f film fused to the tissue.
• Use the joystick to m ove to other areas in the tissue and capture other cells o f interest. • To rem ove the captured tissue, lift the placem ent arm in a sm ooth but swift motion.
W ith the cap in place, lift and rotate the placem ent an n to the unload platform.
• Low er the placem ent arm onto the unload platform - dropping the cap into the extraction slot.
Rotate the placem ent arm to the rest position. The cap will be extractred form the arm and suspended in the unload platfonn.
Slide the cap insertion tool onto the unload platform . M ake sure the open end o f the insertion tool faces the suspended cap and the groove fits over the guide rail.
Chapter 2 M aterials and M ethods
Slide the insertion tool down the groove until the cap is engaged. Rem ove w ith cap attached.
Insert cap into 0.5ml reagent tube.
Press dow n firm ly to ensure an even seal.
Continue extraction according to appropriate protocol.