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PostPosted: Sat Feb 09, 2019 12:34 am 
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Hi all, I also sectioned some old Sunflower leaf blocks yesterday to use for staining practice.Not the best tissue, Sunflower leaf is a swine to get looking anything approaching natural as it tends very much to 'shrivel' under processing, but here are some images of tonight's practice session, which if nothing else are colourful! :D :D

A new stain for me, Acriflavin - a beautiful orange stain but it's not very good for you so must be used very carefully indeed! In fact, as lovely as it is I'm a bit scared of it so won't use it much I think..... This is a 2% solutionln sold as Koi-carp treatment!

I tried it with Safranin, which I mordanted with formalin (mordants increase the ability of a stain to 'stick to' the target tissue) and accentuated (accentuators make a stain brighter and more vivid) with Sodium acetate, both of which I added to the Safranin in 75% isopropanol alcohol as my staining solution for red. Yellow stain tend to remove red stains savagely and I tried to mitigate this effect with the added mordant + accentuator, which did actually work and allowed the Safranin to 'hang-on-in-there' as I added the acriflavin after the Safranin.
Anyway, I used these two not particularly contrasting stains just to see what happened really, after all this was a practice session! :D The result was really rather pleasing and the red and orange/yellow seemed to give a good level of detail and differentiation, which I found a pleasant surprise indeed.... Not perfect but quite pleasing. I've no idea if the acriflavin will fade - time will tell of course.....

Some images - the first is a TS through the large midvein of a Sunflower leaf. Sectioned at a point where one lateral vein is in the process of diverging from the main vein, while another on the other side of the main vein has left the main vein and is sectioned separately and obliquely - as the lateral veins are at an angle to the main vein of course and not parallel to it as in Monocotyledonous leaves....
Image

First hint of the huge number and diversity of hairs (trichomes) found on the Sunflower's leaves (and stem come to that).....
Closer-in to the main vein's vascular bundle,
Image

Now for some amazing hairs all over the leaf....
Minor veins are also seen pretty well in this one,
Image

More hairs! :D I'm feeling a bit itchy....
Image

Image

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Best call it a day there, got a little carried away with images! :oops:

One last section, this one's of a Sunflower stem from last March, sectioned transversely across a node of the stem, the point where a leaf stalk is emerging from the main stem's vasculature as a bundle of 'leaf traces', hence the somewhat lop-sided shape of this section. Stained as I remember with Alcian blue and Safranin....
Image

That's really the last one - honest! :D :D

John B.

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PostPosted: Sat Feb 09, 2019 6:56 am 
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Very impressive John. Not only the photographs but also your knowledge about what the photographs show.

Jan.


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PostPosted: Sat Feb 09, 2019 10:04 am 
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I'll be stealing those as well.. :D


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PostPosted: Sat Feb 09, 2019 10:25 am 
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Just some vague thoughts. Acriflavine is another fluorescent dye, in addition to the well known Rhodamine-B. Fluorescence plays a major part in studies of animal (and human) cells. I do not know if the same holds for plant cells. Fluorescence might add higher selectivity and specifity of the staining, and allow even clearer differentiation between celll types, organelles etc. The main potential caveat is, of course, the auto-fluorescence of the plant, from chlorophylls, vitamins and other molecules.

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PostPosted: Sat Feb 09, 2019 11:07 am 
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Hobbyst46 wrote:
Just some vague thoughts. Acriflavine is another fluorescent dye, in addition to the well known Rhodamine-B. Fluorescence plays a major part in studies of animal (and human) cells. I do not know if the same holds for plant cells. Fluorescence might add higher selectivity and specifity of the staining, and allow even clearer differentiation between celll types, organelles etc. The main potential caveat is, of course, the auto-fluorescence of the plant, from chlorophylls, vitamins and other molecules.


Yes, the extremely selective attachment of the fluorescent stains is used a lot in plant study, here's a link to one such technique. Trouble is I don't have the facilities or equipment (or indeed the funds...) to pursue fluoroscopy. A truly fascinating resource though.

John B. :)

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PostPosted: Sat Feb 09, 2019 11:07 am 
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ChrisR wrote:
I'll be stealing those as well.. :D


Go get-em Chris! :D :D

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