Hi Bill & Gekko, you are very generous and
I thank you both for your support and encouragement. It's great to know others are interested in the mysteries of plants - they never cease to delight & amaze me.
The pollen results really came out of the blue for me, the technique is so simple, basically I dropped pollen (well dried but no other processing whatsoever) into the bottom of the moulds I was testing to make the 'classic Tissue-Tek shaped blocks' seen in all the online videos shot in labs.
The grains stayed at the bottom of the mould after pouring the wax (i.e. against the cut-face to be, of the blocks) and were there waiting for the microtome - amazingly they were sufficiently supported by the wax, although it was only encasing and had not of course infiltrated the interior of the untreated pollen, to stay-put and allow the blade to section them! A dreamy result for sure.
The different sizes and the perfection of their geometry (the moulds) makes them very good and virtually a 'slot into the microtome and start sectioning' solution - I love using them, I'm a total convert - they're a far cry from the 2-part split moulds made from overflow-pipe stops that I use for my hand microtome!
Here are a few pictures of a rather tragic wax-block's sections - this block was discarded because it is over-processed and very dry and brittle....
Stretching sections on water's surface at about 40 deg C;
- Sections in the bath..
- ws_stretching_sections.jpg (138.18 KiB) Viewed 6512 times
A 5µ section showing quite fine detail;
- ws_5micron_in_OH.jpg (258.42 KiB) Viewed 6512 times
Not bad considering;
- Sitting on a slide covered in OH
- ws_fast_green_2.jpg (275.65 KiB) Viewed 6512 times
- ws_fast_green.jpg (266.37 KiB) Viewed 6512 times
Here's a pretty good 15µ section in OH;
- ws_15_micron_section_in_OH.jpg (301.42 KiB) Viewed 6512 times
Good for practicing and testing though - I used Fast Green (cellosolve preparation) simply because it saved me the need to move the sections from OH to water for aqueous staining then back to alcohol to dehydrate and ultimately back to Histoclear in preparation for mounting into a resin mountant. Instead I was able to take a 'shortcut to a slide' by going straight from alcohol (which follows dewaxing as a dehydration) to the cellosolve-formulation of Fast green, which stains well in about 10 seconds if the specimen in covered with alcohol first. After rinsing the excess stain with alcohol for about 15 seconds the slide was ready for mounting in an alcohol-miscible mountant (not resin - resin is miscible with Histoclear, the wax solvent) and cover-slipping!
The fast green is a good stain for cell contents as well as other structures, it has made the condition of the cytoplasm (not the nuclei) visible as an aid to assessment of the section, which is pretty good for a tatty old block. Some stomata have been sectioned nicely without damage, as have the vascular bundles - a major improvement this far!
I'll soon have some new material through processing, I'm going to try to achieve a more targeted result now that I have such good equipment to help me - it's time perhaps to move forward from tissue selected because it makes a good practice subject - my next round of tissue processing will be more difficult and hopefully precise (but I'll still include some good practice pieces as a fall-back!)
Back soon with an update - loads to do, loads to enjoy!
Thanks again for your kindness.
