Picric acid substitute in Johansen's Safranin-Fast Green Protocol

[BramHuntingNematodes]

"Picric acid is perfectly safe as long as you remember several things..." well I'm trying to have to remember fewer things, especially where poisonous explosives are concerned, so I was wondering if there was a substitute. There is Sass's protocol, which is basically the same without picric acid, but it doesn't produce as vivid results. I'm having trouble finding out exactly what role the picric acid has in Johansen's procedure. I have come to think of it as a primary or secondary fixative, but here is it described as merely "to cause safranin O differentiation."

Elsewhere I have seen hot baths in citric acid-citrate buffer as a substitute for picric acid in Masson's trichrome and also as the go-to for general heat induced epitope retrieval. I am attempting to use the safranin-fast green combination to distinguish morphology of a longitudinal section of a mushroom cap preserved in FAA. Y'all know how hard mushrooms can be to see sometimes. So, would citric acid-citrate buffer be useful in this case to break open some of FAA-induced crosslinks? Would this idetify new structures or help brilliancy of the staining at all? If so, should this be carried out prior to staining with safranin or, as in the Johansen method, after (I imagine followed by restaining with safranin? I don't know just thinking out loud here). Any experience or thoughts would be welcome!

[abednego1995]

I've never done plant/fungi specimens, but used Safranin-O and fast green FCF for staining cartilage sections fixed in PFA. If it's about the Safranin-O staining intensity, changing the solution pH (I used sodium citrate buffer) would improve things. Higher pH would yield more intense staining, but would become difficult to differentiate, so I suppose you'll need to do some trials to nail the best pH for your needs.

Also, in my case decalcification with EDTA after fixation leached glycosaminoglycans from the tissue and that would lead to diminished staining. Couldn't solve that problem while I was doing that work, but keeping the period the tissue stayed in solution (yes, glycosaminoglycans aren't fixed with aldehydes) short, and keeping it chilled while going through the fix/decal helped a bit.

P.S. I also tried doing the same stain on heat induced epitope recovery sections with citrate buffer, but boiling pretty much nuked it.

Hope this helps.

Cheers,
John

[Micro_UTRG]

I've only done Sass Safranin - Fast Green on Plant tissue sections.

However, I think a mild acid like Acetic acid is enough to differentiate the Safranin. Not that I'm an expert, but I think that Differentiation just refers to the Safranin being de-stained more slowly from lignified tissues or nuclei, etc. In the end It would be de-stained as well.

[mrsonchus]

Hi, yes, definitely, forget all about picric acid!
May I ask what type of sectioning & mount you are making?
That is to say, are you making wax-infiltrated and embedded microtome-cuts for permanent mounts?

The use of PA as a differentiator for safranin means that it is used to remove safranin from the tissue, after the initial staining with safranin. Safranin is a 'regressive stain' and as such tissue stained with it is (intentionally) overstained, then differentiated (in the sense of differentiation between tissue/cell-types in which safranin remains) in this case with PA. The safranin will differentially leave the tissue; lignin is usually the strongest and last to leave...

Safranin is (as is usual with a regressive stain) the first stain to be applied, fast-green the second or 'counterstain'. I use various agents for differentiation, including 'ordinary' deionised water. For safranin (to be followed by fast-green) I use isopropanol in various ratios with DIW, most often however as 'neat' (or as neat as is easily obtained) 95% (nominally) IPA from a company such as Shiny-Hardware-LTD, with whom I have no connection whatsoever of course.

The first step is always from safranin into DIW to remove excess stain (i.e. that which streams from the tissue for a while before slowing to a virtual stop). Sometimes this will be enough to optain the 'look' desired - especially if using safrain as the only stain, in which case it will very often show a beautiful metachromasia - an often overlooked method I think.

If, as is usually the case, far too much safranin remains after the DIW rinses, the next step will be an IPA/DIW series culminating in 95% IPA. From here differentiation may be continued with 95% IPA or, if this doesn't remove enough safranin, with an acidified mixture. I usually use acetic acid, at 1%, or maybe 5%, even 10% concentrations (in IPA not DIW that is). If I still wish to remove more safranin I 'up the ante' (after a neat IPA rinse to remove the acetic acid component) and add perhaps a tiny amount of HCl to IPA - perhaps 10 drops to 200ml of IPA for example, and proceed carefully - not because there's any danger but because HCl removes safranin (and most other stains) extremely rapidly from tissue... The HCl I use isn't neat - it's only 10% - it's this that I refer to above as being added as 10 drops.

Subsequent IPA rinses are needed to prepare the tissue for the application of fast-green, which is extremely easily removed by water (deionised in all references here). With this in mind fast-green needs to be used in a very nearly water-free mix - I personally use between 0.5%-0.1% fast-green in between 85%-95% (nom) IPA. IPA rinses will remove excess FG. The FG is a 'progressive stain' and is therefore used in usually-several apply-rinse-evaluate cycles until the balance with safranin (in this case) is reached.

If the fast green mixture is too strong (the mixture not the result) it will overstain the safranin to give varying shades of bluish-reddish-greenish colour, most of which are far too dense and opaque and may not only look pretty hideous but obscure detail/s....

A good technique for FG over safranin - apply FG fo 5 second intervals, as in the above cycles, until you have built-up as it were the staining you require. Don't try to remove all safranin from all tissue that you don't want it to stain, such as non-lignified cell-walls as this is nearly impossible. There is a point at which the FG will usurp/overstain the safrain in those cells/tissues without a problem, giving the desired result. If IPA at less than about 85% gets to fast-green the water content will very quickly remove it!
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Another method to remove (or even differentiate then complement) stubborn safranin overstaining is the 'yellowsolve' technique - a variation of which I very often use. This uses a yellow/orange stain the usurp the unwanted safranin, and does so very efficiently indeed. There are two approaches to this, one with water-rinse the other with alcohol rinses, the first removes the safranin to varying extents with no residual yellow staining, the second removes the safranin and allows the ywllow stain (I use 'Orange-G' as it happens) to remain - great for tri-stain with alcian-blue, which in the presence of the yellow stain will turn to a beautiful bright green. A full explanation of these techniques would use about 50 pages of post, I hope the above will help though.

[BramHuntingNematodes]

Thanks everyone, and particularly John B. These are all very helpful comments. Yes, the staining will be of paraffinized sections cut by microtome. I will follow this protocol you have outlined to best of my ability. I may also experiment a bit with citrate buffer on some separate sections. I have read somewhere that glass knives only keep for a day, so I will have two to exhaust and these mushrooms will be my only blocks. I should have surplus ribbon!

One last question: after embedding in paraffin, will I need to clear then take the samples all the way back to water for staining? Is some portion of IPA appropriate? My safranin O is a 1% aqueous solution.

[mrsonchus]

Hi, after embedding, sectioning and floating-out (sections onto slides to dry) leave more-or-less vertically with dust cover (I use those plastic seed tray transparent lids) to dry onto slide - between 1-3 days is fine. Too short drying time will result in tissue loss during the stain & mount processes...

The wax is then removed with a suitable wax solvent - I use 'Histoclear', a limone-based xylene substitute. Two immersions in successive containers to 'dewax' sections - first for about 20 minutes, 2nd (in fresh solvent) about 10 minutes is just right. I have two stages, 'dewax-1' and you guessed it - 'dexax-2'. From there into 95% IPA again 2-stages, first of 3 minutes 2nd the same is fine. At this stage the rehydration (in as you ask, preparation for aqueous safranin stain) may begin. Try a sequence of 75%, 50%, 25% IPA in DIW, for about 5 minutes each will be good. Then from 25% IPA to pure DIW, 2-stages of a minute each will be fine.
Now your sections are on the slide, rehydrated to DIW and ready for the safranin!

Easier than it sounds. If you search my posts you'll find a lot of my microtechnique outlined and imaged.... Try this one for a good look at the process...