Tissue Preparation Protocol for MALDI Mass Spectrometric Imaging For greater detail, refer to J. Mass Spectrom.

2003, 38, p 699-708.

Tissue Freezing -After tissue has been surgically removed, freeze it by slow immersion (30-60 sec) into a liquid nitrogen bath. A cyclopentane/dry ice bath can also be used. -The whole tissue can then be wrapped in aluminum foil, placed into a falcon tube, and storred in a 80C freezer until sectioning.

Sectioning and Mounting -Use a small amount of OCT (optimal cutting temperature polymer) to adhere the tissue sample to the cryostat stage. NOTE: Do not imbed the tissue completely in OCT as the polymer is known to suppress ion formation during MALDI. -Tissue is generally sliced to a thickness of 12 m. Tissue can be sliced to any desired thickness, however, as the slice thickness is reduced, the ability to detect specific analytes may also be reduced. If there are any specific requirements, please contact your Demo/ Applications Chemist at Applied Biosystems/MDS Sciex. -The preferred method of tissue section mounting is to transfer the section onto a cold MALDI plate. The MALDI plate should be equilibrated in the cryostat chamber; cooled to ambient chamber temperature generally between -15C and 20C). Paint brushes are used to transfer the tissue section onto the MALDI plate. -The MALDI plate and the tissue section are then quickly warmed allowing the tissue section to thaw mount onto the MALDI plate. This process can be accomplished by placing a finger-tip on the bottom side of the MALDI plate directly under an edge of the frozen tissue, as the heat from your finger thaws the edge of tissue, you move your finger slowly in the direction of the tissue length and it progressively thaw-mounts onto the plate. -You can mount multiple tissues onto the same MALDI plate by repeating the above procedure. NOTE: Do not mount more than 6-8 tissues per MALDI plate. There must be enough area around the tissue to define a rectangular space without overlapping

any other tissue sections. For MALDI imaging, a rectangular window is set up around each tissue section, MALDI MSI is performed over a defined grid within that rectangle. Tissue sections mounted poorly Tissue section mounted properly!

Please do not rinse the mounted tissue sections in anything prior to shipping without first discussing rinsing procedures with your Demo/Applications chemist. The typical Histology rinsing protocols use solutions (ethanol/water) that tend to elute drugs and other small molecules of interest out of the tissue section.

Shipping At this point you can package the tissue plate(s) in a secure plastic holder, the black plastic plate trays that the blank MALDI plates are shipped in can be used for packaging. Sandwich 2 trays with MALDI plates in between, secure the 2 trays with tape. Seal the trays in a ziplock bag, and place into a Styrofoam container with ample dry ice for shipping. Feel free to use your own shipping protocol, but make sure that the surface of the MALDI plate with tissue mounted does not come into contact with any other objects.