zzffnn wrote:John B,
Is a middle ground between the two protocol possible? The enhanced contrast seems to occur mainly at ovary walls (not so much in the egg cells), though egg cell details may need high magnification to show up.
Hi zz' nice to have you alongside.
Yes, that would be a nice tweak to the technique. The elusive egg-cells are quite hard to find, the very dense (and strongly stained) 'inner tissue' seen with each ovule is I think the 'nucellus' tissue that generates the egg-cell proper. The nucellus itself is a real 'stain-sucker' as it were and I think overstained by these protocols for fine-details, although the bi/multi-nucleate nature of them is easily seen.
As a morphological image the protocol is superb, but for high-magnification chromosome examination for example I prefer the contrast of a very specific Haematoxylin and a light application of an acid cytoplasm stain such as Fast-green or even the general and rather pleasant Bismark brown - a venerable old stain that I have experimented with in the past with some nice results also.
Staining is a vastly underrepresented area online and very hard to find specific information about. It seems to me that images (in the sense of what is seen through the 'scope rather than specifically through a camera) land in only a few categories, that of morphology - the overall detail of tissue and organography, and the 'higher magnification' images concerning anatomy and intracellular details such as chromosomes, plastids etc. and I'm sure others. The overlap between them is small I fear in staining protocol terms....
These methods require me to constantly check and re-check staining progress as I work, a single slide may need to be stained and examined 30 times for example as the right (desired) image is pursued.
Each time for example Fast-green needs to be 'backed-off' an OH wash is needed; but to then attempt the same with water for the reduction of Safranin (properly treated as a regressive-stain) will result in the total and fast removal of the perhaps beautifully stained Fast-green! Some stains are rapidly removed by water (e.g. Fast-green) whilst some are rapidly removed by OH (e.g. Gentian-violet), and some are sensitive to both water and OH but not in the extremely rapid manner of the former two stains.
The main protagonists are the acid and the basic stain classes, combined with the 'progressive' and 'regressive' stains, then there is theory that quotes molecular weight as a factor in stain displacement between two stains or more, then of course there's the effect (if any) of pH, together with the actual point of insertion of these factors in the timeline of a protocol being studied or evolved.
Many, many factors interacting, before the actual tissue-type is accounted for - makes for a fascinating area of study! If you really want to be driven bonkers, try getting acid Fuchsine to survive dehydration towards mounting! arrrggghhhh..... phht phhht!
As I work on these methods I'll post some of the ghastly horrors that I produce also - some of them are quite embarrassing!
Back soon as I get a chance.
I've some (hopefully) mature ovules going into infiltration this afternoon, may even be able to cast some blocks tonight, the ovules are tiny and shouldn't take too long to fully infiltrate with wax...
Thanks for your interest and insights old chap - always very useful.
