Which staining?

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Sm33
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Which staining?

#1 Post by Sm33 » Sat Apr 17, 2021 6:24 pm

These slides are from Triarch Company. Can somebofy identify the staining and post the protocol...Notice how the cytoplasm hasn't been washed and is retained (Can you specify how to maintain cytoplasm in the plannt cell??) I want to make m]y slide maker follow the same protocol...

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mrsonchus
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Re: Which staining?

#2 Post by mrsonchus » Sat Apr 17, 2021 8:52 pm

Hi, this is almost certainly the classic safranin + fast-green stain combination. It certainly looks like it, although there's a certain 'greyness' to the cytoplasmic stain here.

Protocol? An enormous subject dependant upon many, many factors - you'll need to have a good look around the internet for that answer. Start perhaps with a search for staining plant tissue with safranin and fast-green - there will be many protocols to consider - best to pick the simplest, with few or no additives to the basic stains....
John B

Sm33
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Re: Which staining?

#3 Post by Sm33 » Mon Apr 19, 2021 9:32 am

Is this Sass' Safranin and Fast Green Staining? What do you do to preserve cytoplasmic content when you stain the cell?

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mrsonchus
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Re: Which staining?

#4 Post by mrsonchus » Mon Apr 19, 2021 10:32 am

Hi, no way to tell if this is Sass' method I'm afraid. However, Sass' method is pretty simple, with no real additives, and does give good results - I've used it hundreds of times for plant tissue.

Essentially Sass' pethod is:
(slides having been de-waxed, cleared and rehydrated to de-ionized water)
1) Stain with 1% aqueous safranin - ignore the oft-quoted 2-24hr duration! This is an order-of-magnitude too long - start with perhaps 2min...
2) Rinse with DIW (de-ionised water) gently for 1-2mins to allow excess stain to come away.
3) Run through dehydration series of increasingly alcoholic mixtures of DIW and OH (alcohol) to 'pure' (95% isopropanol is as pure as you ever need, and are able to buy!) OH.
4) If safranin is too strong reduce or 'differentiate' it with rinses in OH until satifactory.
5) Dip into approx 0.1% fast-green in 05% OH (I actually use 0.125% in 85% OH and apply with dropper for efficiency) for 'units' of 5sec followed by OH rinses (repeat if/as needed - usually once-only).
6) Disregard the carbol xylene or methyl-salicylate/xylene dehydration final phase of Sass - not necessary as the OH will be perfectly fine for ultimate clearing with a xylene-substitute such as 'Histoclear'.
7) Clear (i.e. drive-off OH) with above Histoclear or equivalent.
8) Mount coverslip with resin-based mountant (I use 'Histomount') for a permanent slide.

As far as the persistence of cytoplasm goes, if your processing is good this should be the case anyway - extra steps may be inserted into processing protocol to remove cell-contents prior to staining of desired.

Johansen's protocol includes enhancements to the safranin - which I use as part of a modified Sass-protocol (see my other post re the use of 61°C mp wax), but also requires methyl cellosolve (which is unavailable) as well as methyl salicylate (which may perhaps be substituted with 'wintergrenn oil')....
John B

Sm33
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Re: Which staining?

#5 Post by Sm33 » Wed Jul 14, 2021 2:06 pm

I want to tell my slide maker not to clear off the cytoplasm in the cells...How shall I tell him? Do you know any specific term for it? Should I ask him not to use xylene?

BramHuntingNematodes
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Re: Which staining?

#6 Post by BramHuntingNematodes » Wed Jul 14, 2021 6:00 pm

I have several older Triarch botanical slides and the staining is absolutely superlative. I would show your maker this picture along with your explanation. There is a bit much artistry, from section thickness to fixation to clearing to come to a definitive answer
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

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mrsonchus
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Re: Which staining?

#7 Post by mrsonchus » Wed Jul 14, 2021 8:59 pm

Hi, hmm, sounds as though your slide-maker is either clearing the cellular contents intentionally, to optimize cell-wall deliniation (which is often desired, depending upon the area/tissue under study and the information desired from such a slide). For example a study of cell-division stages involving commonly perhaps onion-root-tip cells, would require that the cell contents are preserved for staining of the chromosomes and cell-plate formation.
On the other hand say a slide made to study the cellular topography of perhaps the zonation of such an aformentioned root-tip, a quite different perspective of the same tissue, requiring in the first case preservation of cellular contents, in the latter their removal, with preservation and indeed enhancement of cell-walls via staining.

Another possibility is that the cellular contents are indeed still present but either unstained or stained but faded back to invisibility - a quick phase-contrast check would tell you if the contents remain.... This is actually quite common also - some stains or tissue sets can be quite troublesom in this respect and a set that left the lab looking perfect can sooner or later become quite deficient in this area - stain-fading for one reason or another - perhaps the tissue, maybe the staining protocol or even exposure to too much light/sunlight since leaving the lab....

The way to (intentionally) remove cell contents is with the use of a bleaching agent after clearing but prior to staining. It may be the case also, given your reference to xylene, that you understand the 'clearing' process using xylene (in fact I always use a limones-base product - xylene substitute - called 'Histoclear) to mean clearing (i.e. removal) of cellular contents. This is wrong but easily thought. 'Clearing' with xylene (or in my case Histoclear) is a stage where the dehydration alcohol (dehydration as in removal of all water traces) is necessarily (for ultimately mounting permanently in a resin-based mountant) removed - in fact displaced or driven-away by the xylene/Histoclear; the 'clearing' refers not to removal of cellular contents, but the removal of alcohol, and the fact that due to the refractive index of the tissue when saturated with clearing agent, the tissue is rendered almost absolutely transparent - very thin or small sections can in fact become invisible!

So, just let your slide-maker know your exact requirements (e.g. your intended use of the slide/s) and the situation - he'll know how to achieve this, or will realize a problem with slide-preparation if there is one.
John B

Sm33
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Re: Which staining?

#8 Post by Sm33 » Thu Jul 15, 2021 11:39 am

BramHuntingNematodes wrote:
Wed Jul 14, 2021 6:00 pm
I have several older Triarch botanical slides and the staining is absolutely superlative. I would show your maker this picture along with your explanation. There is a bit much artistry, from section thickness to fixation to clearing to come to a definitive answer


Can you send the explanation?

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Re: Which staining?

#9 Post by BramHuntingNematodes » Thu Jul 15, 2021 3:31 pm

Notice how the cytoplasm hasn't been washed and is retained (Can you please maintain cytoplasm in the plannt cell??)
Presumably you are tal;king with a specialist. Call on their specialist's knowledge!
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

Sm33
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Re: Which staining?

#10 Post by Sm33 » Wed Jul 21, 2021 3:26 pm

BramHuntingNematodes wrote:
Wed Jul 14, 2021 6:00 pm
I have several older Triarch botanical slides and the staining is absolutely superlative. I would show your maker this picture along with your explanation. There is a bit much artistry, from section thickness to fixation to clearing to come to a definitive answer
I would want to buy Triarch slides at a lesser price. Who do I contact? My email could not reach zthem

BramHuntingNematodes
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Re: Which staining?

#11 Post by BramHuntingNematodes » Wed Jul 21, 2021 6:20 pm

Sometimes they arise on the secondary market. If you want to contact them directly probably you need to call during business hours. Seems really hard to get people to answer email this last year.
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

Sm33
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Re: Which staining?

#12 Post by Sm33 » Thu Jul 22, 2021 4:44 am

I am from India.....And international calls are expensive...

BramHuntingNematodes
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Re: Which staining?

#13 Post by BramHuntingNematodes » Fri Jul 23, 2021 3:20 pm

I am not very familiar with the biological supply houses in India, but I would imagine there must be some very good ones as India is now one of the world's chief optics manufacturing centers. Biocraft, for example, seems to have a pretty extensive catalog. I don't know for certain if they prepare all their own specimens, but they do mention they have a microtechnique lab. Let us know if you find out anything.
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

JGardner
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Re: Which staining?

#14 Post by JGardner » Fri Jul 23, 2021 7:46 pm

mrsonchus wrote:
Mon Apr 19, 2021 10:32 am
(95% isopropanol is as pure as you ever need, and are able to buy!)
Here in the States 99.9% isopropanol is readily available.

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