I will be using a variety of tissue sections, and will need to stain them for a range of time periods up until the (hopefully) maximum saturation time of the tissue that takes the longest time to saturate.
Is this a question about the best HE-protocol or about the characteristics of the dyes hematoxylin (better hemalaun) and eosin?
Mayers hematoxylin for example is used for progressive staining and for 3-5 µm sections 10 min staining are sufficient and then overstaining hardly occurs. So there has to be a kind of saturation. If the hematoxylin-solution is used progressively or regressively depends on the formula of the solution (amount of hematoxylin in relation to the amount of mordant, and also to the amount of oxidant).
Eosin is always used progressively (overstaining and differentiation). So the point of saturation is regularly exceeded.
In histology we want to stain rather fast, so the used hemalaun and eosin concentration is high enough for this purpose.
So the time for saturation depends on the used concentration.....
In the histodiagnostic lab we stain various kinds of tissue at the same time on the same rack. The HE-protocol for all tissues is the same. The main thing is the thickness of the sections. This should not vary too much (eg. 3 µm to 30 µm). Pretreatment like decalcification also weakens the ability of taking up hemalaun. These specimens may need a little bit longer.
I think you should use EE in aqueous solution. This way you can extend the staining times as you want and do follow very long time differentiation in water to remove deposits of dye that are not chemically bound to the tissues.
Hi Maurizio,
Thank you for your help! The only thing is, I am not looking to extend, I am just looking to find out what generally is the maximum time taken for saturation to occur?
Is this a question about the best HE-protocol or about the characteristics of the dyes hematoxylin (better hemalaun) and eosin?
Mayers hematoxylin for example is used for progressive staining and for 3-5 µm sections 10 min staining are sufficient and then overstaining hardly occurs. So there has to be a kind of saturation. If the hematoxylin-solution is used progressively or regressively depends on the formula of the solution (amount of hematoxylin in relation to the amount of mordant, and also to the amount of oxidant).
Eosin is always used progressively (overstaining and differentiation). So the point of saturation is regularly exceeded.
In histology we want to stain rather fast, so the used hemalaun and eosin concentration is high enough for this purpose.
So the time for saturation depends on the used concentration.....
In the histodiagnostic lab we stain various kinds of tissue at the same time on the same rack. The HE-protocol for all tissues is the same. The main thing is the thickness of the sections. This should not vary too much (eg. 3 µm to 30 µm). Pretreatment like decalcification also weakens the ability of taking up hemalaun. These specimens may need a little bit longer.
It all depends on the exact formula of hematoxylin and eosin being used.
There are hematoxilin that reach to color saturation in a minute and others who need up to half an hour.
The eosin is almost instantaneous and can saturate in just 30 seconds in 1-3% aqueous solution.
I recommend you find the precise properties of the stains before the experiment.
Please note Gudruns comments above. It gives an over view of the H&E stains.
But it is not clear what the intent of the original question is. The goal of the stain is to produce both nuclear (H) and cytoplasmic staining (E). This staining should produce clear cut cytology details. If the H is saturated, you will need to de stained until you can see the details of the chromatin. If your E is over stain, the same problem applies to the cytoplasm. If your worry is that you will miss staining the details you want, by understaining, this is not a big problem. Histolabs process many different tissues with H&E stains at the same time. The over staining is not excessive, in terms of time, to achieve optimal staining. (on different tissues). It is true your will need a bit of experience to get your differentiation right. The best way to get experience with your system is to observe your slides before and after differentiation. By keeping records of this, you will fast obtain the results you want.
The same microscopic observations will let your know if your H&E is saturated. Your nuclear staining will be dense and you will not be able to see the chromotin. Your cytoplasmic staining will be blue too. With the E you will see no cytoplasmic detail and cell bounderies difficult to see. So it is also observation of your tissue which will let you know if your have achieved saturation. Then you will be able to determine your times for the purpose you need. ---- Also see Lee Luna's book, Atlas of Artifiacts. It shows a lot of errors and variations of staining found in histology etc. Good luck
I often do H&E staining for nucleus as the following method: 15 - 30 sec H&E (diluted 1:3 with water), 4 washes with water, and 1 min 15 sec in Bluing agent, 4 washes with water. Then I check slides under a microscope. If the color intensity is not satisfactory, I will stain again using the same procedure. Please remember to filter your H&E and bluing agents before each use. Good luck.
There is no such thing as "Saturation time" for hematoxylin/eosin staining but there are two types of hematoxylin formulas: one is called "progressive" and the other "regressive". Mayer's formula is the most common of the "progressive" and Harris "regressive". With Mayer's you place the section in it and check it until it reaches the staining you desire that will vary with the type of tissue and section thickness. It is time consuming and somewhat cumbersome to proceed and, as a general rule, it will take longer to stain. With Harris you will stain for, at the most, 5 minutes and the tissues will be strongly stained. After that you will "decolorize" the stained tissue until you get the desired staining intensity. That "decolorization" or "regression" is equivalent to what you do with iron hematoxylin in the step known as "differentiation" with different strengths of iron alum. I prefer Harris over Mayer but in staining the researcher can choose. Summing up: there is no general "time", there is no "saturation" and in your case I would use a regressive formula, such as Harris.
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