TITLE: Sample preparation protocol for cross-sectional microscopy of hair fibers
AUTHORS: Tina Lasisi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to prepare hair fibers for cross-sectional microscopy. </div><div class = "text-block"><span class = "embed-element"><iframe width="560" height="315" src="https://www.youtube.com/embed/OSgiErHmiDE" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture" allowfullscreen></iframe></span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/cfb789f892fdf2a45ab1bc1f046932df431a7708165393e0f13fab2c9267c0eb/crtxbbavx.png" /></div></div>

[STEPS]
?. [Embedding Hairs ]
The following steps are for embedding hair fibers into the plastic chips.
For embedding you will need:hair samples2 prepared plastic chips per sample2 tweezers/forcepsa hot plate heated to   isopropanol for cleaning
Prepare hair sample for embedding. Take 3-5 hairs per sample and cut samples down to 5cm if they are over this length.
?. [Cutting Hairs]
Prior to cutting, the embedded chips must be cooled for at least 2hrs.
Cutting the hairs will require:PanaVise with blade attachment (see comment)plastic bags for storing the embedded chips
Cutting tools
Cutting the hair samples requires a sharp blade. Our lab uses the PanaVise 502 Precision PanaPress with cable cutting attachment. 
A sharp razor or other implement may work, but the blade must be sharp enough and it must be applied with enough pressure. Also, this must be done with minimal contact with the sample as body temperature will heat the embedded sample causing the hair to shift while being cut.
?. [Cutting Hairs]
?. [Preparing plastic chips for embedding]
The following steps are for cutting and thinning the (polycaprolactone) plastic to smaller size for embedding
Polycaprolactone plastic melts at   . To produce small, thin chips for embedding, you will need:A sheet or strip of plastic  cut into 2x2cm squaresA hot plate (heated to   )3 metal blocks to use as weights ScissorsSquares of parchment paper (5x5cm)Heat resistant gloves
?. [Imaging]
For the imaging, you will require:a clamp or stand holding the sample upright (see materials)a microscope capable of magnifying to 10x
?. [Preparing plastic chips for embedding]
Have adequate protection for your hands. The metal will be too hot to move with bare hands - use heat resistant gloves.Heat plasticHeat one of the metal blocks on the hot plate for 5mins. Place square of plastic (with parchment paper underneath) on hot plate for 30 sec.
?. [Preparing plastic chips for embedding]
Flatten plastic.Cover heated plastic with square of parchment paper.Place heated metal block on top for 30 sec.
?. [Preparing plastic chips for embedding]
Cool plasticRemove plastic (with parchment on either side) and flatten between two cold metal blocksLeave to cool for 5 mins.Peel off parchment and remove thinned plastic chips.
?. [Preparing plastic chips for embedding]
Cut plastic to desired dimensions using scissors or scalpel.
The appropriate size will depend on your hair samples. We recommend 20x10mm for most hairs and 20x5mm for very short hairs. Anything smaller will be challenging, though not impossible, to section.
?. [Embedding Hairs ]
Place one prepared plastic chip on a parchment paper on the heated block and wait until the chip is translucent (about 30sec).
?. [Embedding Hairs ]
Once plastic is translucent, grab a hair fiber with tweezers on each side and stretch over the plastic, pressing the hair into the plastic so it is completely submerged. Continue until all hairs for that sample are embedded.
Embedding the hairsThe hairs should be should be stretched taut over the plastic. In the process, some hairs may snap, so having spares is recommended. 
Take care that all hairs are laid parallel to each other to ensure perpendicular cuts for the entire sample.
?. [Embedding Hairs ]
Heat second prepared plastic chip until translucent.
?. [Embedding Hairs ]
Carefully overlay the second chip over the first (containing the embedded hairs).
?. [Embedding Hairs ]
Apply metal block overtop to flatten sample for 15 sec.
?. [Embedding Hairs ]
Remove the embedded hair sample and place between two cool blocks and leave for 2mins.
?. [Embedding Hairs ]
When cooled, peel off parchment paper, trim excess hairs and plastic into a rectangular chip.
?. [Embedding Hairs ]
The sample should now be stored in a cool room at  for at least 2 hours.
4 °C
?. [Cutting Hairs]
Place chip under press and cut the sample in half. The sample may also be cut using a single blade.
Work in small batches and work quickly as the chips will heat up. You have the option of cutting the samples in half (resulting in two mirroring sections) or you can trim the top of a sample to expose the cross section.
?. [Cutting Hairs]
Immediately store the samples in cold room or freezer until imaging.
?. [Imaging]
Mount the hairs vertically.
?. [Imaging]
Image each hair in the embedded sample individually.
Take care not to leave the samples under the microscope unattended, as the lights may melt the sample.
?. [Imaging]
Store immediately in cool room or freezer.