TITLE: Matrix Sublimation via In-House Developed Sublimation Apparatus
AUTHORS: David       Anderson, Martin Dufresne, Jeffrey D. Messinger, Jamie Allen, Katerina V Djambazova, Angela  Kruse, Christine A. Curcio, Kevin L. Schey, Richard M. Caprioli, Jeff Spraggins

[DESCRIPTION]
Scope:
This protocol describes the use of an in-house developed sublimation device to deposit small molecule matrices onto tissue sections for high resolution MALDI imaging mass spectrometry (IMS). Concentrations will vary based on matrix type.

[STEPS]
SECTION: Matrix Preparation
11. Dissolve 5mg of matrix in 2 mL of acetone for a total matrix concentration of 2.5 mg/mL.
 

SECTION: Sublimation Procedure
16. Place apparatus lid on the base, ensure that base is fully seated in the indentions on the induction heater top cover. Start pulling vacuum.
SECTION: Sublimation Procedure
17. Once it is clear that vacuum is established, ensure that bath drain stopcock is closed. Add a small amount of acetone (20 mL) into the well than fill with dry ice and allow to cool for 3 min. 
SECTION: Sublimation Procedure
15. Check that mating surfaces of the lid, base, and the O-ring are clean, and carefully seat the O-ring in the groove in the base.
SECTION: Sublimation Procedure
14. Place matrix pan on the heat plate in the apparatus base.
SECTION: Matrix Preparation
12. Place matrix pan on a level surface and evenly distribute the matrix solution by dispensing the solution in an equilateral triangle around the center of the matrix pan.
 

  

SECTION: Matrix Preparation
13. Wait for solvent to dry (3 min).
SECTION: Sublimation Procedure
18. Wait for the pressure to fall to < 50 mTorr measured at the pump (usually ~25 mTorr). 
Note the pressure at the pump and at the apparatus.
SECTION: Sublimation Procedure
19. Turn on internal thermocouple. 
Turn on induction heater, set heater to 390F temperature setting, and start a Start 10 min timer.
 

SECTION: Sublimation Procedure
20. Turn off the heater and remove dry ice chips from the ice bath. Replace ice bath with warm water and open to drain excess water. Repeat warm water exchanges until the apparatus is at room temperature.
 

SECTION: Clean Up
22. Clean matrix pan and sample holder with acetone.
SECTION: Cold Trap Preparation
5. Ensure the cold sleeve is at least 2/3 full of ethanol or isopropyl alcohol when trap is fully seated.
SECTION: Cold Trap Preparation
6. Carefully add dry ice chips to the ethanol bath in the sleeve and allow chips to sublimate off. 
Repeat until minimal sublimation of dry ice is observed (ethanol has reached equilibrium temperature).
  

SECTION: Sublimation Apparatus Set up
7. Ensure that the vacuum line is securely installed between the cold trap and the apparatus lid.
SECTION: Sublimation Apparatus Set up
10. Place sample holder in the lid using the embedded magnets.
  

SECTION: Sublimation Apparatus Set up
9. Place sample slides in the sample holder indentations. 
Use copper tape or magnetic holder as needed to secure samples as close to the center of the holder as possible.
SECTION: Sublimation Apparatus Set up
8. Ensure that the electrical connections between the heater controller and the passthrough in the apparatus base are secure and remain dry.
SECTION: Annealing
23. Place samples on a heating plate (130oC) for 30 seconds.
SECTION: Sample Section Preparation
2. Wash sectioned tissue slides with three separate solutions of 150 millimolar (mM) ammonium formate for 45 s each.
SECTION: Sample Section Preparation
1. Chill three solutions of 150 millimolar (mM) ammonium formate on ice for  30 minutes.
SECTION: Sample Section Preparation
3. Dry tissue using a stream of nitrogen gas.
SECTION: Sample Section Preparation
4. Store in a vacuum desiccator for 20 min .
SECTION: Sublimation Procedure
21. Turn off the vacuum pump and break vacuum with the leak valve on the vacuum manifold and remove matrix-coated samples.