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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This method details our microtomy (sectioning) process for reasearch specimens involved with HuBMAP.</div><div class = "text-block">This process follows the microtomy process that follows after receiving, processing, and embedding of research specimens into formalin fixed, paraffin embedded tissue blocks. </div></div>

[STEPS]
?. [Tissue Block Preparation]
Ensure the tissue blocks you have chosen to section have been correctly embedded, 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)
?. [Microtome Preparation]
Locate your tissue flotation bath / water bath and fill it 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.
?. [Slide Preparation]
Be sure to locate the glass charged microscope slides that are appropriate for the tissue type or project you are about to perform microtomy for.Wear gloves for this step to keep the slides clean of debris/prints, and to prevent ink/graphite from smearing.
?. [Tissue Block Facing]
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]
Trim gently into the block to expose the tissue surface to a level where a complete representative section can be cut.
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 your 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 from the melting ice pieces or ice water will also make the tissue easier to cut.
?. [Performing Microtomy]
After your tissue blocks have chilled on the ice tray and are very cold to the touch, they should now be suitable for precision sectioning. Remove your first block from the ice tray and secure it into the microtome's block chuck.
?. [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 condensation from the frozen block accumulate near the knife holder, 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 sections is 4-5 µm.- It is normal to have to scrap the first few sections that appear on your ribbon due to holes or other cutting artifacts.
?. [Performing Microtomy]
After your ribbon has begun to form on the front of the knife holder plate, use a pair of forceps (or whatever your preference is for transferring ribbons from the microtome to the water bath) 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 water bath.
?. [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 section.
?. [Performing Microtomy]
After your paraffin ribbon has floated on the water bath for approximately 20-45 seconds, use your forceps to select the section you wish to keep.
?. [Performing Microtomy]
After selecting your section, find the appropriately pre-labeled microscope slide that matches the tissue block you sectioned.
?. [Performing Microtomy]
Dip the slide into the waterbath, and place adjacent to and slightly under your chosen section.
?. [Performing Microtomy]
Adjust the section as desired on the slide with the forceps, and lift upwards out of the water bath, as shown in the clip below.
?. [Performing Microtomy]
Place your newly made slide into a slide drying tray or into a slide staining rack, as pictured below.After completion, let your slides air dry overnight or dry in a 50°C oven for 1 hour.
?. [Cleanup Area]
After you have sectioned your last tissue block, unlock the microtome knife blade holder's lever/spanner.Using a magnet or forceps, carefully remove the microtome blade and place into the waste container on the blade's box, or in a 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, water bath 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 water bath and dump the remaining water into your nearest sink.
?. [Cleanup Area]
Clean the emptied water bath with warm water and dawn dish soap, then replace to the water bath's bench unit.