May 08, 2020
Cutting FFPE Sections for Imaging Mass Cytometry
- 1University of Florida;
- 2University of Zürich
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: Jeff.spraggins@vanderbilt.edu
Protocol Citation: Marda Jorgensen, Michelle Daniel 2020. Cutting FFPE Sections for Imaging Mass Cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.bfz4jp8w
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: May 05, 2020
Last Modified: October 02, 2020
Protocol Integer ID: 36636
Keywords: HuBMAP, TMC-Florida/Zurich, Imaging Mass Cytometry
Abstract
FFPE block are cut into thin sections.
Materials
MATERIALS
FFPE block
Rotary MicrotomeThermofisherCatalog #HM355S
FFPE Block Cooling UnitCatalog #TES99.430
Stretching table
Deionized Water
Microtome Blade
Brushes
Glass Slides Superfrost 25x75x1.0mmThermo Fisher ScientificCatalog #10.0344.01
Safety warnings
The microtome blade is very sharp, care must be taken to avoid injury.
Cooling the FFPE Block
Cooling the FFPE Block
1.1 Cool down the FFPE block
a. Switch on the cooling device and set it to 4°C
b. Put the FFPE block into the cooling device.
Switching on the microtome
Switching on the microtome
1.2 Prepare the microtome
a. Connect the cooling unit in the back with the cable and switch it on at the
front.
b. Switch on the microtome using the button at the back of the machine.
c. Install the water bath with the filter.
d. Add deionised water to the water bath and lower the compartment until the
grid is slightly covered with water.
e. Place a fresh blade in the blade holder.
f. Check the water flow. If water is not flowing rinse the tubing with a syringe
filled with water.
g. Set the water flow to 3 or 4.
h. Fix the precooled FFPE block to the specimen clamp holder.
i. Adjust the angle between blade and FFPE block by unlocking the clamping
lever and then adjusting the orienting screws accordingly.
j. Set the trimming (Trim) and cutting (Feed) thickness by pressing the top knob
once or twice, respectively.
k. Unlock the wheel with the mechanical hand wheel brake on the right-hand
side underneath the wheel.
1.3 Switch on the stretching table
a. Switch it on and set it to 37°C.
Cutting
Cutting
1.4 Trimming
a. Always use a new sharp blade.
b. Trim carefully and make sure that your cutting angle is fine, if needed readjust.
c. As soon as you think you are cutting tissue, take up a cut and have a look
under the microscope.
d. Exchange the water bath before starting cutting sections that you intend to
collect.
1.5 Cutting
a. Cut in a uniform and gentle rotation.
b. Let the cut slide down into the water bath.
c. Wait until the cut is flattened before taking it up on a glass slide.
d. Wipe the glass slide dry on the bottom side before placing it on top of the
stretcher.
e. Exchange water on a regular basis if sample produces a lot of paraffin debris
and between samples.
Switching off
Switching off
1.6 Switching off the microtome
a. Block the wheel to avoid any injuries as soon as you are done cutting
b. Dispose of the blade
c. Stop the water flow by turning the wheel down to 0.
d. Discard the water, clean the water bath, filter and grid, place them to dry.
e. Clean the water slide.
f. Clean the microtome from paraffin debris.
g. Switch off the microtome at the back and disconnect the cooling block.
h. Switch off the FFPE Block cooling device.
Section drying and storage
Section drying and storage
1.7 Drying sections
a. Dry the sections overnight or for 1-2 hours at 37°C. Do not extend the drying
for too long.
1.8 Storage of FFPE sections
a. Store all FFPE fresh cut and dried sections at -80°C or stain them within a week.