Dyed In The Wax!

[mrsonchus]

Hi all, here's an interesting subject that I stumbled across online some time ago, and now have the capability to try with a chance of some meaningful results, now being able to rely on good-sectioning and thus remove section-integrity as a surely complicating factor...
The method is of staining a section while it is still in wax, that is to say it has been sectioned and dried onto a slide in the usual way and is ready to be dewaxed with Histoclear. At this stage the slide (with section firmly dried-onto it of course) is simply put into a stain, in this case I've used aqueous 0.05% w/v 'TB0' AKA Toluidine Blue for a time (between about 2 & 10 minutes)...
At the end of this time, the slide is washed, gently as usual, in DI water and left to air-dry until all water has gone - about 2 hours I've found at room-temp.
The section, if stained and dried like this without the wax still supporting and saturating the tissue, would suffer horrible damage and be rendered useless, but the wax seems to completely prevent this and the tissue appears to be perfectly stable and intact after drying.
Now the slide can be dewaxed with Histoclear for 10 minutes (seems to be optimal for just about all of my sections) without agitation I'd recommend, maybe with a final couple of gentle 'dunks' but no more and mounted with a resinous medium such as 'Numount'.
You'll notice that no movement from dewaxing to water (via a series into and a series out of alcohol as conduit between Histoclear and aqueous-staining) has been necessary! Effectively straight from section as cut to staining the straight to dewaxing and mounting! Wow - that's what I call a shortcut! Much potential tissue compromise/damage is also completely avoided too!

Time surely for less talk and more pictures!
Here are a couple of sections stained in this manner - the first attempt!

This is the trusty Carex.pendula leaf XS stained with TB0 in the wax;

Looks pretty well-stained...

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This looks pretty good, a really pleasant surprise for me as I really didn't know what to expect... The contrast looks very good and there's definitely a good differentiation of tissue-types, due to the metachromasia afforded by the TB0 stain presumably..

This one's a section of root from a garden 'weed' (or indigenous species of British wildflower as I prefer to think of it! ) called a Sonchus - a type of 'Sow-Thistle'... I've been looking at the manner in which lateral-roots originate from deep within the main root of a plant, said therefore to be 'endogenous', and this series of sections has given me some very good observations of this I think - the origin of the new (lateral) root is clear to see, even without dewaxing!

Amazing to think this section is still in the wax-section...

ws_sonchus_asper_endogenous.jpg (459.33 KiB) Viewed 5218 times

A little ragged perhaps, but this section is from a tough root and was cut without any of the softening and hydration that I would usually apply immediately before sectioning (standing wax-block cutting-edge-down in ammonia for a few minutes - a tip given kindly to me by Naphthalene) - but overall I'm pleased with it at first-peek!

The proof of this colourful test will be in the dewaxing.... should be intersting!

This is the root after dewaxing and mounted permanently in resinous 'Numount';
I love this section, the lateral-root is sectioned longitudinally and therefore both XS and LS may be examined within the same section!

Sectioned at 8µ - a good thickness I think..

ws_W82_8mic_sonchus_root.jpg (198.79 KiB) Viewed 5217 times

Here are a couple of the Carex-leaf after dewaxing and mounted in the same way as the root..

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ws_x20_SA01_carex_2.jpg (293.68 KiB) Viewed 5217 times

Looking good so far - go to go, back soon with more..

[gekko]

Beautiful work and very nice images.

[mrsonchus]

Thanks gekko, that's very encouraging and kind.

[zzffnn]

Indeed, I like your professional-looking images! Very beautiful too!

[mrsonchus]

Indeed, I like your professional-looking images! Very beautiful too!

Thank you!

The deployment of the metachromasia of TB0 is a very simple and effective technique it seems, especially the wax-staining method, very fast results and very good tissue-protection! It's like cheating with counterstaining..... but I like it!

[mrsonchus]

I've had a read of some details of the metachromatic-staining technique (with TB0) and it seems that the blue areas are lignified (i.e. 'reinforced and strong) cell-walls and the purple-ish tissues are non-lignified. Looking at the distribution of colour in the images it seems to make perfect sense too, the outer (epidermal) surfaces/layers may be expected to be 'tougher' than the inner tissue, and the clearly visible region on the upper-epidermis around the 'inside' of the 'hinged keel' area that I suspect may be bulliform is as expected non-lignified and therefore more mobile than the rest (blue regions)...

I may be quite wrong about the bulliform area but even if I am, it's very encouraging to see the clearly-defined metachromatic differentiation actually happen on one of my own slides rather than seeing it in a book!
Fascinating to finally see these details as read about in the books! I'm having a great time, thanks to, you guessed it - The Mighty Shandon!

[mrsonchus]

Here is the gnarled-looking (tap) root of the Sonchus seen sectioned above...

The root of the investigation

ws_700x525_sow_thistle (12).jpg (119.74 KiB) Viewed 5201 times

I had a go at a bit of dissection and hand-sectioned a few thick 'slices' (XS) for processing and ultimately an encounter with the microtome. While doing this I noticed how the 'outer layer' came cleanly away from the 'core' leaving the lateral-root as part of that core... interesting.

This is what started me off with my interest in what I discovered was called 'endogenous root formation';

What's going on here?

ws_700x525_sow_thistle_root (5).jpg (39.6 KiB) Viewed 5201 times

Guess what's coming next!

ws_root_wax_block.jpg (187.45 KiB) Viewed 5201 times

Giving some promising sections..

ws_root_in_wax_on_slide.jpg (117.12 KiB) Viewed 5201 times

The start of an adventure! A bit of reading and quite a lot of work gave me this beauty;

Lignified tracheal-elements are blue..

ws_700x525_root_1.jpg (157.56 KiB) Viewed 5201 times

Interesting to see effectively the cross-sectioned main tap root and the longitudinally-sectioned lateral root. 2 - views for the price of 1! Notice in the previously posted lower magnification picture how the tissue layout of the lateral root is the same as that of the tap root too (as expected but nice to be able to see never the less..

This root still has a lot to offer, the walls of the tracheal elements have a fascinating structure when seen at the higher end of the magnifications - I'll post some higher-objective pictures to show this as soon as I get a minute, may even be time to fire-up the 100x! What fun from and old root!

Back with further explorations soon.

[billbillt]

Hi John,
More wonderful and interesting work!....
BillT

[mrsonchus]

Hi all, I've had a go at re-orientating, trimming and re-embedding a couple of root parts originally intended fot the cross-sections seen above. I trimmed them parallel to the lateral-root (along each side of it, across the main tap root whence it originated..

Here's the orientation in the mould that gave me the earlier cross-sections;

The original orientation

ws_700x525_100_3830.jpg (81.97 KiB) Viewed 5177 times

Trimmed to this;

Trimmed ready to turn up the other way..

ws_700x525_100_3831.jpg (83.99 KiB) Viewed 5177 times

Then 'turned upright' to present the longitudinal aspect to the microtome's blade;

Fiddly even with a dissecting 'scope, but easily done really.

ws_700x525_100_3839.jpg (36.79 KiB) Viewed 5177 times

Here's a couple of pictures, sectioned, stretched onto the wonderful Leica positively-charged slides and dipped (very carefully - they're still wet!) into Toluidine Blue stain for about 30 seconds before again very carefully rinsing and standing vertically to drain - not dry when these pictures were taken but I wanted a quick idea of how the orientations were looking...

Here's a complete LS right across the root;

Blue - lignified, purple - non-lignified tissue

ws_700x525_x4_root_long.jpg (99.08 KiB) Viewed 5177 times

This one seems to show the emerging lateral-root causing a 're-routing' of the main tap root's vascular tissue;

Is this the start of a lateral-root?

ws_700x525_x20_root_long_stack.jpg (123.29 KiB) Viewed 5177 times

These seem quite close to the orientation I'm trying to achieve, that of along the lateral-root as it emerges from within the tissue of the main root - these sections aren't down to this area yet, further depth should see the lateral-root appear...

Not bad though considering I've quickly stretched them onto a slide then dunked them rather unceremoniously straight into a flask of aqueous Toluidine Blue and hoped they stayed on the slide (slides being positively-charged may have helped) before going straight to the 'scope with them still wet to fit in a few pictures tonight! Looking very promising indeed.

I've another quick post following this one with a few extra pictures...

[mrsonchus]

Here are a few more of the quick pictures taken from tonight's LS root-sectioning attempt;

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ws_700x525_root_long-0005.jpg (114.36 KiB) Viewed 5176 times

And a quick look at the stem belonging to the owner of the root XS - Sonchus.asper;

ws_700x525_slide_12_sonchus_asper_stem.jpg (92.75 KiB) Viewed 5176 times

Going well, should get these dewaxed and mounted tomorrow, and hopefully I can 'drill down' with the Shandon to the exact plane for the section along the elusive lateral-root, what fun!

Oh yes, the stem XS was akso stained in the wax, but using Safranin, which has worked equally as well as the first-tried TB0 did!

Back soon, bedtime calls..

[gekko]

More excellent work and beautiful pictures.

Back soon, bedtime calls..

Considering the amount of work you do, and the care and craftsmanship you devote to it, and then the time to prepare the posts (pictures and detailed description) I find it very hard to believe that you set aside any time at all for sleep.

[mrsonchus]

Aha gekko - that's very generous of you, thank you. I've always got time for a good sleep - one of my favourite hobbies! Pleased you're enjoying the investigations..
Thanks for your interest and complement.

p.s. I'm looking forward to taking some images of the dewaxed and mounted sections, they should be good, considering how well the preparations still in the wax seem to perform - just goes to show I think that contrast is (nearly) everything in microscopy, the stains are pleasing to the eye, but the contrast and therefore edge-detection, for both camera and our own eyes I suspect, that they provide, may well be the key factor for resolution of details - the ultimate goal (for me personally) although the sheer beauty of some folk's pictures is a joy to behold too!
Who would have thought roots could be so interesting!