Washing Protocol for Intact Proteoform MALDI on Human Kidney

While the tissue is within the vacuum desiccator, pour all necessary solvents and solutions into the Coplin jar. These have a volume of approximately 75mL and to reduce the variability in extraction fill the jars consistently.

Prior to submerging the tissue section in solvent and solutions, ensure that the timers are set beforehand. While three timers are not necessary, it vastly improves the process.

If any metadata is present on the slide written in permanent marker remove it at this time. Take a photo and note within a notebook prior to removal, this is necessary as this will contaminate the Coplin jars within all washes.

Submerge the tissue section within a Coplin jar filled with a solution of 70% ethanol for 0h 0m 30s

Immediately move the tissue section with tweezers to then next coplin jar containing 100% ethanol for 0h 0m 30s

After completion of the tissue fixation steps, immediately move the tissue section with tweezers to then next Coplin jar containing Carnoy's solution for 0h 2m 0s

Carnoy's solution is a 6:3:1 volumetric solution of ethanol: chloroform: glacial acetic acid, while other solutions could be put within plastic staining jars a glass Coplin jar is highly recommended for this and other polar aprotic solvents.

Note: dipping, agitation, and any movement of the slide within the Coplin jar will dramatically change outcomes of all washes. Tissue is prone to detach from the surface within this step.

Following exposure to Carnoy's solution, submerge the slide with 100% ethanol for 0h 0m 30s

After completion of the lipid depletion washes, submerge the tissue sections within nanopure water with 0.2% trifluoroacetic (TFA) acid for 0h 0m 15s

Note: dipping, agitation, and any movement of the slide within the Coplin jar will dramatically change outcomes of all washes. Tissue is prone to detach from the surface within this step.

After exposure to water, submerge the slide within 100% ethanol for 0h 0m 30s

Ensure this is a new solution of 100% ethanol not used within previous steps.

After the completion of all steps, dry the tissue for 0h 0m 30s under a diffuse stream of nitrogen, take care to ensure that liquid does not pool within the surface. The tissue section is now prepared for the next steps within the tissue preparation protocol.

Discard all solvents and solutions properly, and best practice is to use fresh solvents and solutions for subsequent washes.