Hi all, I've been working with the
double-embedded tissues some more and have made some pleasing progress;
In my last post the tissues were inside their agar-blocks and ready to face the perils of the rest of a complete histological cycle for the ultimate goal of being able to section the
tissue and the agar whilst they were 'double-embedded' inside a normal paraffin-wax block, mounted on the mighty Shandon in the usual way as any 'normal' tissue embedded only in paraffin, as I've been doing before this, my first ever trial with the double-embedding technique with the goal of being able to precisely (accurately at least...
) handle and orientate very small tissue such as anthers, or tissue that as a consequence of it's 'floppiness' tends to simply fall-over when attempting to get it to for example 'stand on edge' during 'normal' wax embedding, with a view to cutting sections across perhaps a leaf-blade...
Sounds too good & powerful a technique to be true I keep thinking, but as it turns out, this first trial, using commonly available chef's agar bought from a food-store (the packet's instruction simply said 'mix as you would gravy'!), has proceeded almost without problems... The agar/tissue-combi' was from this point on treated as if it were just a usual single-piece of tissue like for example a piece of stem.
Below is a pasted excerpt from my notes, (I use the superb and
free MS 'One-Note' for my notes now) saves me extra typing...
----------------------from my notes------------------------------------------------------------------------------
Double-embedding, tissue pieces that are very difficult to orientate when embedding may be 'double embedded' to make their handling and orientation when embedding into wax easier and far more precise. A very useful technique for samples that are tiny, e.g. flower anther or that are prone to falling over when attempting to get them to 'stand up' e.g. leaf blade, as they are being embedded in molten wax in order to present the correct cutting edge to the microtome knife when mounted in the wax block on a microtome.
Tissue pieces that were already fixed for more than 48hrs in FAA were available and therefore used in this (first) trial. The pieces were 4 in number, 2 pieces of Sonchus.asper leaf-blade intended for XS and 2 halves of a Sonchus.asper's stem that had been spilt lengthwise for the intention of taking LS.
These pieces were cast into agar and left in fridge at 4deg for 3 days. Whilst the tissues themselves had been thoroughly fixed, the agar itself had undergone no fixation whatsoever before the tissue pieces were embedded in it.
The agar pieces were taken from the fridge and trimmed to convenient sizes and shapes that would enable the precise and desired orientation of the tissue that they contain to be achieved when they are 'stood' in the correct position in a T-Tek mould during wax embedding.
Processing of these trimmed agar/tissue blocks was then carried out as if the whole blocks were comprised only of tissue, the agar acting and reacting to chemical and physical stresses suffered during processing in the same way as the fixed tissue that they contained…
Next the agar/tissue blocks were put into 75% IPA as a start of dehydration, the concentration being a good match for that of the FAA fixative whence the agar-embedded tissue came.
The following is the protocol used:
Saturday 8pm - into 75% IPA to start dehydration
-------overnight--------------
Sunday 11am - into 95% IPA (1)
Sunday 1:30pm - into 95% IPA (2)
-------overnight--------------
Monday 2:30pm - into 1:1 Histoclear (HC):IPA mix to start clearing of IPA from tissue
Monday 9pm into pure HC
------------overnight------------------------------
agar had turned totally transparent to the extent that it was completely invisible - this suggests complete infiltration into the agar by HC clearing agent…
Tuesday 11:30am - into molten wax at 60-62deg (1) as start of infiltration
Tuesday 2pm - into wax (2)
Tuesday 7pm - cast 4 wax-blocks, labelled T1,T2,T3 and T4
Tuesday 9pm - roughing carried out on microtome of 2 blocks
All 4 blocks in fridge
-------------------overnight-----------------------------
--------------------------notes end-------------------------------
Here are a few pictures;
Here's the start of dehydration - tissue is sinking after the application of vacuum (using a rubber-bung, a fish-tank airline and valve and a syringe to draw-off air from glass container..) to OH,
- ws_sinking_agar.jpg (113.43 KiB) Viewed 5383 times
This is a wax-block cast with agar/tissue embedded,
- ws_double_embedded_block.jpg (116.58 KiB) Viewed 5383 times
Here's a view back-lit to show block's contents better,
- ws_backlit_double_embedded_.jpg (70.3 KiB) Viewed 5383 times
The following pictures are not of the above pictured stem-pieces but of another block containing a leaf-blade piece... It seemed an inferior block to the one pictured above so I risked my first-go using it instead of the superior (stem LS) block just in case I cause complete chaos with my first attempt!
Sections from 'roughing' - not final quality, just quick tests to see if the principle is sound - please excuse poor quality
Here a couple of (appallingly rough
) sections are stretching on water (de-gassed) at 44deg;
- ws_agar_stretching_1.jpg (105.37 KiB) Viewed 5383 times
Here they are (sorry again - they're not the finished article...), floated onto a slide..
- ws_agar_on_slide_1.jpg (120.42 KiB) Viewed 5383 times
The division seen in the agar is an artifact from my earlier (i.e. just after initial agar-cooling) meddling in order to release a pesky air-bubble that had crept into the agar - during agar-embedding, though the wax has 'taken up the slack' and filled in the split, thus completing the encasement and embedding of the tissue/agar - this didn't occur in the other blocks, it's a consequence of my clumsiness rather than a failure of the procedure of double-embedding, my own stupid mistake I'm afraid, however it was not sufficient reason to abandon the block so I used it as intended! (that's my excuse anyway
).
These trials so far are making the technique look to have enormous potential, carried out properly (after I've had a lot of practice!
) it offers a hugely effective, useful, efficient and above all relatively easy technique for the orientation of all manner of tiny or just plain awkward tissue-types and shapes.... I'm looking forward to taking some better sections, maybe tomorrow if I get time, and 'finishing off' the process by drying, dewaxing, staining and mounting these examples - a hugely interesting and enjoyable trial, I've already learned a lot from this attempt so far - can't wait to see it to it's conclusion.....
Musing - If the clearing-agent (in this case Histoclear) didn't destroy the agar during clearing of the OH, will it be able to dewax the slide and remove the agar also, or is there another step needed to do this - hmmmm I wonder what's going to happen in a few days when the slides are dried and ready for dewaxing!
Perhaps because (in these sections) the thickness is onnly 8µ the agar will be removed in the same manner and at the same time as the wax... I certainly hope so - onward-ho! There's only one way to find out!
Having a great time, back soon with hopefully some rather better sections, these were a rush-job and only taken with my 'roughing' blade - nowhere near as good as my current 'best cut' blade! (honest!
).
Back with more soon, and some more staining adventures are to come probably tomorrow on my other topic thread! See you there!
..