Fixation Protocol for Fresh Frozen Tissue Samples (post-MALDI)

After the tissue has been imaged via MALDI IMS, the slide should be placed in a coplin jar or glass petri dish with a solution of room temperature 4% paraformaldehyde in PBS (*must be room temperature).

Place jar/dish on a shaker for 5 minutes.

Discard the PFA (the MALDI IMS matrix will come off the slide in this step).

Add fresh 4% PFA in PBS and shake for 5 more minutes (for a total of 10 minutes in PFA).

• Washing with PFA solution will fix and rehydrate the tissue as the matrix is removed. This preserves the tissue and antigens, and significantly reduces artifacts.

Add fresh PBS for 3 minutes on shaker.

Repeat this wash two more times for a total of 3 times for 3 minutes each.

• pH should be neutral

Incubate the slides in 50 mg/mL bovie serum albumin (BSA), 10% (v/v) host-derived serum (depends on which antibodies they use), and 0.5% (v/v) Tween-20 in PBS for 30 minutes.

Be very careful. Tissue will be very fragile.* This permeabilizes the tissue slowly.

• The Tween cannot be substituted for triton X, as triton X is too harsh and will degrade the tissue.
• 30 minutes works for most. Can do up to 2 hours before the tissue gets very damaged/adherence issues/weird background.

Add fresh PBS for 3 minutes on shaker.

Repeat this wash two more times for a total of 3 times for 3 minutes each.