Jan 31, 2023
Fixation Protocol for Fresh Frozen Tissue Samples (post-MALDI)
- 1University of California, Davis;
- 2Vanderbilt University
Protocol Citation: Elizabeth Neumann, Jamie Allen, Jeff Spraggins 2023. Fixation Protocol for Fresh Frozen Tissue Samples (post-MALDI). protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l276d3g1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 05, 2023
Last Modified: October 18, 2023
Protocol Integer ID: 74843
Keywords: Cell DIVE, MALDI IMS, fixation, matrix removal
Abstract
Scope:
To provide a method for removal of MALDI matrix, fixation and preparation of tissue sections for Cell DIVE.
Guidelines
Special Notes:
1. Make sure no tissue drying, particularly after matrix removal.
2. Glass petri dishes can be used for more fragile tissues.
3. This protocol can be used on Cell DIVE fresh frozen samples.
Materials
Materials:
1. ITO slides with tissue from MALDI experiment
2. Coplin staining dish, Fisher S17495
3. Glass petri dish, Fisher 08-747C
4. Tabletop shaker
Reagents:
1. 4% paraformaldehyde in PBS, Fisher AAJ61899AK
2. Phosphate Buffered Solution (PBS), Fisher 70-011-044
3. Bovine Serum Albumin, Fisher BP671-10
4. Normal Donkey Serum, Jackson ImmunoResearch 017-000-121
5. Tween 20, Sigma P9416
6. Milliq ddH2O
Solutions:
1. 1x PBS
100mL 10x PBS
900mL ddH2O
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1000 mL total solution
2. Block Solution:
2.5 grams of BSA
5 mL of reconstituted donkey serum (reconstituted in 10mL of ddH2O)
0.25 mL of Tween 20
44.75 mL of 1X PBS
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50 mL total block solution
Safety warnings
Paraformaldehyde should be used inside a chemical fume hood.
For research use only.
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.