A New Batch of Paraffin-Blocks

[mrsonchus]

Hi all, I've managed to get a fresh set of specimens processed over the last couple of days, and have just taken a few tentative sections, following roughing...

I processed some stem and root pieces from good-old Sonchus.asper (AKA 'Prickly Sow Thistle'), and also used the last few pieces of Aloe-leaf XS that I've had sitting in FAA (fixative) for a couple of months and that have been sectioned quite a few times already, but never with any particularly good results unfortunately.

The protocol used was a shortened-version which I thought I'd give a try, especially as the tissue-pieces I'm now able to prepare are so much smaller and more precisely taken, thanks to my purchase of an excellent stereo-zoom dissecting 'scope some while ago now. One unexpected problem was that the little £5 (Chinese-made) coffee-warmer that I use to keep my metal wax-moulds warm died this morning and was therefore unavailable, although this didn't make a lot of difference in practice thank goodness.

The root-material has always been difficult and seems to be (perhaps unsurprisingly) tough and prone to drying-out and becoming quite brittle with the usual long protocol (that takes anything up to about 5 days!). The hope being that the shorter-protocol/s attempted this time, actually 2 slightly different versions, one with a shorter dehydration-phase, the other with a shorter clearing-phase, would go some way to lessening this problem, although I may of course have picked the wrong factor to alter for a remedy to this problem.
I've tried standing the tissue-blocks in cold ammonia to soften and hydrate the tissue immediately before sectioning but have had limited success with this technique, although it definitely helped (a tip kindly given to me by Naphthalene)...

Here are the 4 blocks I have cast from this run's tissues;

ws_700x525_blocks_labelled.jpg (50.23 KiB) Viewed 2646 times

This is the brief and very shortened & simplified protocol and a variation labelled 'FAST TRACK' on my whiteboard, that was used for the above wax-blocks;

ws_700x525_tissue_protocol_12_sept.jpg (102.18 KiB) Viewed 2646 times

Here are some pieces of fixed stem (longitudinally-cut) and root-pieces (that I attempted to 'clean-up' a bit better than the last time I attempted root-sections) sitting in fixative about to enter dehydration...

ws_700x525_specimens_clearing.jpg (60.73 KiB) Viewed 2646 times

The blocks and blades were kept on a frozen block from the freezer, with the option of placing into the glass-container atop the cold-block that contains water to help hydrate the block-face a little as an aid to sectioning difficult material such as the root-tissue. I colour-code the blades, as a set is needed, one for 'roughing' (starting off the block and making sure it's cutting straight and true) moving into the block in relatively large steps until tissue starts to surface in the sections - these I colour red, the 'next grade' of blade, one that has had some use and is usually fine for say 10-15µ sections of un-problematic tissue (if such a thing exists! ) I colour-code black, and finally coloured green blades are virtually new and in very good condition - a totally new blade I will colour with 2 green dots until It's cut say 50 sections, when I 'downgrade it' to the one green dot... This simple system works well and is unambiguous.

ws_700x525_DSCN2176.jpg (105.04 KiB) Viewed 2646 times

I only had time to start with a few quick sections of root last night and have taken a quick picture of one through the stereo 'scope to show in this post - it can be seen to have sectioned pretty well, without breaks in tissue-interfaces as were seen in earlier root-sections between the inner 'core' (vascular cylinder) of the root and it's outer (parenchymatous cortex) tissue. Usually my sections have divided (i.e have a gap in them) at the interface of these 2 tissues - the intriguing casparian-strip lies here...
Here's a quick picture 'in the wax';

ws_700x525_SAT1_slide_3.jpg (76.14 KiB) Viewed 2646 times

Looks promising, as soon as I've cut some hopefully half-decent sections and dried and dewaxed them I'll post some details following their subsequent progress through the tortuous stages of staining and mounting - it's a multi-stage and long detailed process but very interesting and rewarding when a fine stained & mounted slide comes out of the other end (hopefully that is )..

Back soon with some section-pictures and some extra detail regarding problems that have arisen - these brief protocols are definitely somewhat limited, and the processing-stages - this could turn into a long series if I'm not careful....

[billbillt]

Hi John,

Can't wait to see more... Very interesting...

BillT

[JimT]

Looking forward to your results.

As I said you are working on a very precise project that I would never attempt to do.

JimT

[mrsonchus]

Thanks Jim and Bill, results in ASAP, I'm stretching some sections onto slides this evening, may be able to finish a few tomorrow if they dry well.. Having a great time!