Blake,

If I am understanding your question correctly, I would suggest that you envision your sample as though it were a loaf of bread. Take slices of the "bread" and place them in the cassette the way you would place them on a plate if you were making a sandwich (so you cut the slices and turn them 90 degrees so that they lie cut side down in the tray). You can fit a few slices of your sample in one tray. Each slice should be no more than 4 mm thick.

If you are interested in seeing sections throughout the length of the sample (from start to finish) you'll need more than one tray. Or, perhaps you just take a few "slices" from the entire sample (beginning, middle, and end) and reserve the rest of the sample for another time (or discard when you are absolutely certain you no longer need it).

Make a note of the orientation of each slice in the tray as well as where each originally came from in the sample. (So, if you make five slices you might draw a small diagram of your sample and use dotted lines to show where you cut, then number the slices 1,2,3,4,5...then draw another diagram of your histology tray(s) and mark where each slice 1,2,3,4,5 (1,2,3 in one tray, 4,5 in another or whatever you desire).

This should be done prior to processing in order to allow ease of cutting. You can use lens paper or biopsy pads to prevent your samples from curling during tissue processing.

Hopefully I understood your question correctly - and these suggestions might help you.

This is like the problem of sectioning mammalian retina which opens up like a flap. For paraffin sections, we used to prepare the tissue by embedding midway in a block, and process it into a block. Then we would turn the block perpendicular and cut ribbons. This works even in frozen sections since after freezing you can readily turn the frozen block and reimbed with additional solution and cut the ribbons.

The problem we faced with frozen sections of retina was that the cross sections being very small, do not readily adhere and tend to wash off during staining, which may also happen for you depending on the consistency of the tissue. For this we used to prepare slides coated with optically clear albuman, teat it with glutaraldehyde and accept the section on these such that the sections are cross-linked to the substratum. It worked well for us.

To get specific areas like fovea, if you have any comparable issues, The initial dissection of the retinal falp used to ensure by keeping track of orientation throughout the procedure. Once you get the hang of it it became routine.

One addition to Melissa. When you embed the cut skin-pieces that are rather thin and about 3-4 mm high, it may happen that they fall down flat again.

So let the bottom of the mold with the molten paraffin cool down for a second with the skin-piece in place and right orientation. "glue" it on the bottom. This works well for 3-4 pieces in one mold. Orientate them longitudinal /parallel to your cutting-direction of the microtom, so that the knife hits the skin on the narrow site. In the end it looks like 3 small walls of skin in the block.

There are two ways to solve the problem, (1) You can embed the skin with the skin fall down flat, then take note of the direction of the long axis of the skin so when you are doing the blocking you place the embedded specimen on the wooden block in that the skin will stand vertical on the wooden block. (2) Pour your melted paraffin wax in the ice tray hold your skin vertically in the melted paraffin and hold it until paraffin has solidified considerably then allow it to set completely. Take note of the orientation so you will not miss the orientation when you are doing blocking.

Histologically, a "cross-section" of skin = 'orthogonal section' and/or 'section plane' (= 90° vertically with regard to epidermal surface)

to display in stained sections all levels of skin from epidermis to subcutis/subcutaneous fat (instead of "bread slices" (:-)) )...

cf. enclosed image of prepared ~1 mm slices from human skin for LM as well as consecutive/correlative transmission electron microscopy (TEM).

NB: cornea (despite really thin sort of "membrane") and skin (specs in diagnostic histology spanning about 20+ times, in CLEM (correlative LM&TEM) 7-10 times the 'orthogonal' thickness of cornea), in your case, human skin equivalents (~20 mm in diameter, ~ 1mm in thickness) for better precision during 'trimming' small orthogonal slices for processing and (paraffin) embedding should be observed with the aid either of 'magnifying goggles' / 'surgical/medical loupes' or by using a dissecting "microscope=stereoscope". Always prevent any surface drying during "trimming" (done best in your case with a (properly) de-greased new razor blade, so use at least physiological saline if not (better) appropriate buffer solution (correct pH and tonicity) to wet all surfaces properly until transfer to the primary fixative (if your skin equivalent has not been fixed initially as a whole....).

NB: I have done a lot of corneae as well as skin [also human skin equivalent culture slabs] specimens for correlative (LM-EM)-microscopy and always prepared a bit thicker orthogonal cornea and skin slices for paraffin embedding of those for comparative histological studies.

It might be tricky to have a clue about the final "location" of one slice proper in a paraffin block /embedding cassette containing 3 or more consecutive slices in case you have to section the whole skin equivalent slab QUANTITATIVELY (dimensional sketches / accompanying drawings always to be made adopting /adhering to previously settled conventions), but colleagues in their former replies have indicated solutions for that problem. If you need only some sections for 'control' or 'demonstration' of morphology, or even IHC and other special purposes out of the whole hum. skin equivalent slab it might suffice to divide (with a new) razor blade the whole circular slab into 3 zones and to mount these positioned upright on to the (still cooling wax zone at the ) bottom of the embedding cassette.

Hi, if you can do a cross section, you can not make a photo under the microscope and show them, so you have to split the skin into smaller pieces.

As I understand correctly your question, its very simple to section the tissue cross-sectionally. First of all, the skin should be embedded in the paraffin wax on flat as we do for longitudinal sections, but without cassette on top. After cooling, skin specimen with paraffin wax should be taken out and we can see the embedded skin tissue. The paraffin block can be trimmed as much as we like to handle the block containing the tissue and we can re-embed the correctly oriented skin block fast into the molten paraffin wax in mould kept on the cooling chamber so as to avoid the melting of the paraffin block with the cassette on the top of it. This way we easily embed the skin tissue to cut it cross-sectionally.

Agree with@Gudrun Lang.

Regards

To best represent a flat specimen, embed it on its edge and make a larger amount of slides, using step sectioning, or just skip directly to your region of interest.

To embed:

1) Fill your mold with molten embedding paraffin (60-63 degrees Celsius).

2) Place the mold on a cooling tray.

3) Use heated forceps to quickly place your samples equidistantly on their edges at approximately 45 degree angle to the direction of future sectioning. This will "glue" the tissues in place and prevent them from falling over.

4) Place a cassette on top of the mold/tissue if using cassettes.

Keep track of your tissues as you embed them. For best results, make a separate block for each one. Cool slowly to prevent cracking. Placing the blocks into a refrigerator shortly after embedding and letting them cool is the best practice.

If you have a larger tissue, but are dead set on keeping it intact, you may use a larger mold. While working in eye pathology, I used custom made steel molds for thicker larger specimens: a thicker piece of sheet metal and two pieces cut off of a steel angle stock. Those two L-shaped pieces could be placed together on top of the sheet metal and make any size of a block you wished.

The best histology protocols I ever found were in AFIP Manual. ISBN: 188104100X. Check out chapter six.

A permanent link to the manual https://hdl.handle.net/2027/mdp.39015029715979