TITLE: HuBMAP - Tissue Sectioning for CODEX Specimens
AUTHORS: Leigh Propper, Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This method details the microtomy process for specimens that will be stained and analyzed using the Akoya CODEX® system.</div><div class = "text-block">This process applies to paraffin embedded blocks containing tissue of no more than 1cm x 1cm in size.</div><div class = "text-block">FFPE tissues for CODEX® analysis must be sectioned onto prepared poly-l-lysine coated coverslips.</div><div class = "text-block">Cut and mounted tissue sections can be stored at 4°C for up to one(1 )month prior to staining.</div></div>

[STEPS]
?. [Microtome Preparation]
Fill  flotation bath dish completely with de-ionized or purified water.
?. [Microtome Preparation]
Turn the flotation bath on, and set the water temperature to 42°C.
[Flotation Bath]
At this step, also inspect the microtome you are planning to use; Ensure it is clean, well maintained, and set at the correct angle.
?. [Tissue Block Preparation]
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]
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.
?. [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.
Before putting a microtome blade into the knife holder, BE SURE to utilize the locking lever under the flywheel.
?. [Tissue Block Facing]
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).
?. [Tissue Block Facing]
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.
?. [Tissue Block Facing]
Notate all pertinent information on the CODEX Microtomy Tracking Sheet .
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.
?. [Tissue Block Facing]
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.
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.
?. [Tissue Block Facing]
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.
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]
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.
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.
?. [Coverslip Preparation]
Use an aerosol spray to clean coverslips from dust and lint prior to use.
?. [Performing Microtomy]
Remove your first block from the ice tray and secure it into the microtome's block chuck.
?. [Performing Microtomy]
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.
?. [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.
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.
?. [Performing Microtomy]
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.
- 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.
?. [Performing Microtomy]
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. via GIPHY
"Clip of sectioning a spleen by MJ @ University of Florida"
?. [Performing Microtomy]
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. via GIPHY
Small clip of moving a paraffin tissue ribbon to waterbath by MJ @ University of Florida
?. [Performing Microtomy]
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.
?. [Performing Microtomy]
After selecting your section, use the specialty CODEX forceps and VERY carefully pick up the corner of the poly-l-lysine coverslip.
?. [Performing Microtomy]
VERY gently, dip the poly-l-lysine coverslip into the waterbath, and place adjacent to and slightly under your chosen section.
?. [Performing Microtomy]
Adjust the section as desired on the slide, and gently lift upwards out of the waterbath.  via GIPHY
"Clip of a tech carefully selecting a poly-l-llysine coverslip and picking up a section for CODEX staining. Note the specialized staining rack."
?. [Performing Microtomy]
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
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.
?. [Cleanup Area]
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).
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.
?. [Cleanup Area]
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.
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. Example of Paraguard
?. [Cleanup Area]
Carefully remove the glass dish from your waterbath and dump the remaining water into your nearest sink.
?. [Cleanup Area]
Clean the emptied waterbath with warm water and gauze, then leave it upside down to dry.