Dec 20, 2019
Version 2
HuBMAP - Tissue Sectioning for CODEX Specimens V.2
- 1University of Florida
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: Jeff.spraggins@vanderbilt.edu
Protocol Citation: Leigh Propper, Marda Jorgensen 2019. HuBMAP - Tissue Sectioning for CODEX Specimens. protocols.io https://dx.doi.org/10.17504/protocols.io.basdiea6
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 in our group and it is working.
Created: December 20, 2019
Last Modified: April 02, 2020
Protocol Integer ID: 31269
Keywords: histology, codex, microtomy, tissue sectioning, tissue, pathology, research specimens, histopathology, hubmap
Abstract
This method details the microtomy process for specimens that will be stained and analyzed using the Akoya CODEX® system.
This process applies to paraffin embedded blocks containing tissue of no more than 1cm x 1cm in size.
FFPE tissues for CODEX® analysis must be sectioned onto prepared poly-l-lysine coated coverslips.
Cut and mounted tissue sections can be stored at 4°C for up to one(1 )month prior to staining.
Guidelines
- Managers and supervisors - are responsible for making sure that technicians are properly trained and equipment and facility are maintained in good working order.
- Laboratory personnel - are responsible for reading and understanding this SOP and related documents and to perform these tasks in accordance with the SOPs.
Materials
MATERIALS
Water
KimWipesFischer Scientific
Shandon™ Cartilage Curved Thumb Forceps, Curved, Fine Point, standard, 5 in. (12.7cm)Thermo FisherCatalog #1631TS
Flotation BathFisher Scientific
Microtome Blades (Thermo Scientific Ultra)Thermo ScientificCatalog #3053835
Gauze 4x4 Non-Sterile SquaresFisher ScientificCatalog #MSD-1400249
Microtomy BrushCancer DiagnosticsCatalog #SLK1000
MicrotomeDiagnostic PathologyCatalog #HM 315 / HM 325
Fisherbrand™ Superfrost™ Plus Stain SlidesFisher ScientificCatalog #22-034979
EZ-QUIK SLIDE STAINING RACKFisher ScientificCatalog #NC0103846
Tissue-Tek® Cold PlateVWR ScientificCatalog #25608-942
Moist Mark Plus Slide/Cassette MarkerCancer DiagnosticsCatalog #SKU: MP2100
Dumont Forceps (Cover Slip Forceps)Fine Science ToolsCatalog #11251-33
EMS Cover Glass Staining RacksFisher ScientificCatalog #Catalog No.50-949-581
Safety warnings
- Use physical safety precautions and extreme care when working with sharps (disposable blades).
Before start
- Wear gloves at all times when handling human derived tissue blocks and sections.
- Ensure you have proper slides, blades, forceps, and your personal preference of gauzes/wipes.
- It is recommended that bent angle tipped forceps be used to handle the poly-l-lysine coverslips.
- Use EXTREME CARE with the poly-llysine squares for mounting the CODEX tissue. Any nicks or dings in the mounting coverslip will make it immediately unusable for this process.
Microtome Preparation
Microtome Preparation
10m
10m
Fill flotation bath dish completely with de-ionized or purified water.
5m
Turn the flotation bath on, and set the water temperature to 42°C.
42 °C Flotation Bath
Note
At this step, also inspect the microtome you are planning to use; Ensure it is clean, well maintained, and set at the correct angle.
5m
Tissue Block Preparation
Tissue Block Preparation
- The best practice for most all facing (trimming) and sectioning is to place a few paper towels or a Wypall on top of the cold plate tray, and dampen it with water.
Cold Plate Example: Sakura Tissue Tek Poly Cold Plate
Note
- Ensure the tissue blocks you have chosen to section have been correctly embedded (1cm x 1cm), and all wax has been scraped/cleaned off the sides of the block.
- Ensure the tissue blocks have been correctly labeled with proper identification (case #, type, etc)
Coverslip Preparation
Coverslip Preparation
If the supply of the coated poly-l-lysine coverslips for CODEX microtomy is getting low, additional slips must be made ready.
Please follow the protocol below if you are close to expiration on or running low of CODEX coverslips.
CITATION
Tissue Block Facing
Tissue Block Facing
After locating the appropriate blades to use for your microtome, carefully select a blade from it's container, and place it inside the microtome's knife holder area. Be sure to secure it in place with the knife clamp/lock spanner.
Microtome blade dispensing from storage box.
Note
Before putting a microtome blade into the knife holder, BE SURE to utilize the locking lever under the flywheel.
10m
After securing your knife and double checking your microtome settings one final time, retrieve a block from your ice tray and secure it in the microtome's chuck (block holder).
A set up, tidy microtome, with a secured knife in the knife holder and tissue block in the microtome chuck.
3m
After the block is secured, use the coarse advance wheel on the left side of the microtome to carefully, approach the block with the blade and cut a few thin sections to ensure the positioning is correct. Adjust if necessary.
Small view of the Codex Microtomy Sheet.
Note
The process of sectioning CODEX specimens requires specific documentation at the tissue block trimming and microtomy stage.
As you begin to cut into a new or already used tissue block, you must keep track of how many microns of tissue are removed from the block during trimming and sectioning.
Attached is a generic document that is used to keep record of what tissue block is being cut. All identification factors, trimming data, what tissue sections were used and their purpose, as well as storage information.
Formal documentation of how far into the tissue block you have traveled as well as how many sections were used is an extremely important and necessary function for this project.
Trim gently into the block to expose the tissue surface to a level where a complete representative section can be cut.
Record the amount of tissue removed and discarded.
Note
Trimming is normally done at a thickness of 10-30 µm. This can be performed with the advance flywheel alone, or a combination of both the advance and coarse advance wheels using the rocking method.
After each tissue block has been trimmed to expose a representative surface of the specimen, place each block back on the wet lined ice tray. Let each block chill for approximately 10-15 minutes.
Note
Cold wax allows thinner sections to be obtained by providing support for harder elements within the tissue specimen. The small amount of moisture that penetrates the block will re-hydrate the tissue providing a better section.
Coverslip Preparation
Coverslip Preparation
- Prepare a clean, dry surface near the microtome, and place your poly-l-lysine dish with the coverslips there.
- There is no good way to mark or identify these slips due to the nature of the processing on CODEX. Therefore, it is extremely important to pay attention and keep track of what you're cutting and where you place it after sectioning.
Note
Wear gloves for this step to keep the tiny slips free of smudges, and to keep from contaminating ANY part of the container the slips are kept in.
poly-l-lysine coverslips secured in a petri dish with coating and expiration date labeled.
10m
Use an aerosol spray to clean coverslips from dust and lint prior to use.
Performing Microtomy
Performing Microtomy
Remove your first block from the ice tray and secure it into the microtome's block chuck.
1h 30m
If you used the current exposed part of the microtome's blade on trimming, be sure to slide the knife over to a new, clean section before performing microtomy.
Using your coarse adjustment wheel on the left, adjust the block holder to be as close as possible to the edge of the knife, but not touching it.
Note
A helpful tip: When you adjust your chuck holder towards the blade and begin to see small water droplets from the chilled block accumulate on the blade, stop using the coarse advance wheel.
Very carefully, unlock and rotate the hand wheel on the right side of the microtome so the block holder is moving up and down in a steady, even manner.
A ribbon of wax and tissue should begin to form down the front of the metal plate covering the knife holder.
Note
- - Double check the micron thickness setting prior to this step. Normal thickness for CODEX sections is 5µm.
- - It is normal to have to scrap the first few sections that appear on your ribbon due to holes or other cutting artifacts. Do not collect imperfect sections. Remember to keep an accurate record of all tissue cut from each block.
After your ribbon has begun to form on the front of the knife holder plate, use a pair of forceps (or whatever tool you prefer) to gently grip the bottom of the ribbon and another pair of forceps to gently grip the top of the ribbon nearest the blade.
Gently pull the ribbon up and away from the microtome and towards the waterbath.
A selection of tools ideal to use for securing and floating tissue ribbons during microtomy.
Lifting a ribbon of complete sections away from the knife holder plate.
Note
"Clip of sectioning a spleen by MJ @ University of Florida"
- As carefully as possible, shift the ribbon of tissue sections toward your waterbath, and then quickly but gently lay the tissue out on the bath in a "dragging" type motion (either gently towards you, or gently away from you).
- Wait a few moments while the tissue sections sit on the waterbath, as the heated water will help expand any compression and remove some of the wrinkling or folding in the ribbon.
Preparing to place a paraffin ribbon of tissue sections across a waterbath prior to creating a slide.
Small ribbon of tissue sections afloat on a waterbath - note they are very nice, with no wrinkling, folding, or knife lines.
Note
Small clip of moving a paraffin tissue ribbon to waterbath by MJ @ University of Florida
After your paraffin ribbon has floated on the waterbath for approximately 20-45 seconds, use your forceps or tool of choice to separate the sections from each other.
Keep section order in mind for record keeping purposes.
After selecting your section, use the specialty CODEX forceps and VERY carefully pick up the corner of the poly-l-lysine coverslip.
Dumont #5/45 - Cover Slip Forceps
VERY gently, dip the poly-l-lysine coverslip into the waterbath, and place adjacent to and slightly under your chosen section.
Adjust the section as desired on the slide, and gently lift upwards out of the waterbath.
Note
"Clip of a tech carefully selecting a poly-l-llysine coverslip and picking up a section for CODEX staining. Note the specialized staining rack."
Place your tissue containing coverslips into the specialized metal staining rack and let your slides air dry overnight. Following the air drying process, the sections must be stored at 4 °C for no more than a month, in a compartmented box where the sections cannot be stacked or damaged and where their location can be tracked.
4 °C
Specialized custom staining rack appropriate for the poly-l-lysine coverslips.
Note
As there is no way to mark these types of slides with identification, it is best to keep one case per rack / tray for organizational and accuracy purposes.
1d
Cleanup Area
Cleanup Area
10m
10m
- After you have sectioned your last tissue block, lock the microtome advance wheel and release the microtome knife blade holder's lever.
- Using a magnet or forceps, carefully remove the microtome blade and place into an approved waste container (sharps container).
Note
It is a good practice to clean your microtome if you are done for the day. This prevents paraffin gunk buildup on your microtome, waterbath and floor area, in addition to being ideal for safety.
Using a microtome brush, brush away all remnants of tissue and paraffin around your microtome, it's catch tray, chuck and behind the knife holder.
Note
A commercially prepared product known as "ParaGuard" can assist with the removal of wax and provide a more efficient microtome clean up. It should be lightly used and vigilantly wiped off with gauze after use.
Carefully remove the glass dish from your waterbath and dump the remaining water into your nearest sink.
Clean the emptied waterbath with warm water and gauze, then leave it upside down to dry.
Citations
Step 4
Franchesca Farris, Marda Jorgensen. Poly-Lysine Coverslip Preparation
https://protocols.io/view/poly-lysine-coverslip-preparation-baeribd6