Eosin proper concentration for histology?

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Raul
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Eosin proper concentration for histology?

#1 Post by Raul » Tue Oct 27, 2015 8:40 pm

As the title says I want to know what the suitable concentration of Eosin would be for most histological applications. I checked on the internet and some proposed 1% other 0.5% and even 0.2% (of course the staining time differed with each concentration). I would wish to know from someone that had good results with a particular concentration and maybe if he/she knows the advantages and disadvantages of the other concentrations.

Thank you, Raul.

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mrsonchus
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Re: Eosin proper concentration for histology?

#2 Post by mrsonchus » Thu Oct 29, 2015 5:42 am

Hi Raul, can't give any specific advice I'm afraid, I haven't used Eosin but; what I can tell you is that when staining it's most often useful to think in terms of 'percent-time' (that is, the product of the two), e.g. 0.5% for 30 mins = 15 '%mins' as does 1% for 15 mins...
This simple relationship seems to be very reliable in my experience, remember though that it works with all other factors being equal, such as tissue-type etc, like must be compared to like.

Having said that I find with certain stains (the hideously temperamental 'Orange-G' springs to mind) that below a certain concentration they just will not 'take' - presumably a concentration-gradient (or the generally relatively small molecule-size of the yellow stains maybe) of some sort is at play then. As a result of this I have several of my permanently-mixed and available stains in 2 or 3 strengths, for example I use stain such as Methylene blue or TBO at a 0.05%aq concentration, whereas to try to use Orange-G below 1% will drive you crazy! :)

I use the lower % such as 0.05% when I would like to give a stain plenty of time in the staining (I use Coplins) jar, a stain such as Safranin will really 'get a good grip on the tissue' as an aid to differentiation this way, after which a fast & 'strong' stain such as Fast green (in cellosolve usually) can bludgeon the tissue for about 10 seconds!

So - as a possible answer to your question, start with 0.5% and see how the tissue reacts. Can you give more detail of what the tissue is, what other stain/s are involved (Romanowsky?) - presumably you're using an aqueous preparation?

I don't know if eosin is a 'regressive stain' like safranin, if it is then overstaining is the best way, with a careful and gradual removal of unwanted stain from the tissue with a suitable solvent, water is good for clearing messy excess stain from the swamped slide but to 'start eating into' the stain attached to the tissue until you've only stained the desired cells I often find a weak acidic-OH works superbly.

The only way to perfect the method for your particular tissue is by a series of trials of different 'percent-times', you'll also learn much more than just enough to deal with the one scenario as a bonus!
The longer times at lower % will usually be far more timing-tolerant, any staining time below about 30 seconds (yes, Fast green can make you panic!) can be so sensitive to time that it become unreliable - you've only to be unable to reach something as you're working to completely ruin the process for example! (don't ask me how I know this - please.... :oops: )..

But, longer times (over say 10 mins), especially I find with aqueous staining, sometimes cause tissue to start feeling the need to leave the slide's surface - which is very bad! :)
No reliable 'one-size-fits-all' protocol I'm afraid that will work for all tissue and processing types, try a few protocols, make notes of everything, as a poor result with one tissue may be a good result with another..

Tell us what you try and what the results are if you've time , it will be most interesting and helpful.

Sorry to be somewhat vague and imprecise, staining is a steep and long learning-curve, I've been on it for about 6 months now and I've only recently managed to get good results with Safranin & Fast green counterstaining!

Good luck, let us know how you get on. :)
John B

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Raul
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Re: Eosin proper concentration for histology?

#3 Post by Raul » Sat Oct 31, 2015 7:44 pm

Thank you for your reply John B, it seems that 1% at 2 min does the job well compared to the rest. Thank you for your advice on Safranin and Orange G it may come in handy at later times :).

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mrsonchus
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Re: Eosin proper concentration for histology?

#4 Post by mrsonchus » Sat Oct 31, 2015 10:46 pm

Raul wrote:Thank you for your reply John B, it seems that 1% at 2 min does the job well compared to the rest. Thank you for your advice on Safranin and Orange G it may come in handy at later times :).
You're welcome Raul, that protocol sounds just about right and is very similar to one I use for TBO (the same in fact) with sections at between about 5 ->12 µ.

Glad you're sorted. :)
John B

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