This modified alcoholic Eosin Y includes the addition of stabilizers for a prolonged shelf life. It is intended for use in the histologic demonstration of cytoplasm.
Erythrocytes, collagen and the cytoplasm of muscle or epithelial cells should stain with three different intensities of pink. This Eosin-Phloxine has been modified with the addition of stabilizers to prolong shelf life.
A highly stable bluing solution, which is designed for bluing hematoxylin stained nuclei. This solution can be used for both paraffin and frozen sections. It is non-toxic and odorless, available in ready-to-use or concentrate. Slide A here represents a good section from the block.
We should be able to take Slide A, place it over the top of that embedded tissue sample, and they should match identically in size. There you know that you are giving the pathologist the best representation you can of that piece of tissue. Remember the red areas that I showed you on the map?
Here is more. After staining, there are unstained areas in the tissue; there are areas with eosin staining, but no hematoxylin staining. This a really cool one that I kind of want to stop and talk a little bit about, because I had this happen to me.
There were these little round dots on my slide that I thought, what is this? I turned to a professional, an expert in this field, and I said, what is this? So if you ever see these perfectly round pieces of unstained except for eosin in your tissue, you know you've got paraffin on your slide. Staining the tissue nuclei with the primary stain, hematoxylin, it may involve differentiation, it may involve a bluing step.
It probably will involve counter-staining, dehydration, and clearing. Xylene is a popular substance to use. There are xylene substitutes. The second one will have a little carryover. The third one will be nice and clean to make sure that all the paraffin has been removed.
If you do decide to use a substitute, then please maybe put an extra one in, maybe leave it a little bit longer, and for sure change it a little more often than you would your xylene. Xylene removes any remaining paraffin left on the slides or in the tissue. At this point in time, the sections, if you hold the piece of tissue up and look at it, or hold the slide up, you should see that the tissue is actually transparent; you can hardly even see anything is on it.
Next comes hydration. If you make the cells upset by throwing them into water too quickly, that could just throw off your entire stain. Basically I believed for a long time that this was just stepping down so I could make my cells happy when they got to the water at the end.
When we get to the water and all the xylene is gone, then we want to remove all of the alcohol from the slides also by washing them well in running water. Absorption just means a solution passing through another substance until they reach equilibrium. Hematoxylin is absorbed by the cell like spray perfume is absorbed by the skin.
If we were to mix that up with some water and put a slide in it, nothing would happen, but when we combine it with what we call mordant, which is a number of metallic salts, and we combine our hematoxylin with a mordant, it because an extremely strong nuclear stain. I want to explain something to you on this slide. Truthfully, hematin is the result of hematoxylin dye meeting with aluminum salts, whether it be aluminum, iron, or chromium, and resulting in this great nuclear stain. It is connected by what we call covalent bonds.
A counterstain is just a secondary dye, in this case eosin that is used to enhance the primary dye without interfering in it. Adsorption, not absorption, is a physical reaction dependent on the charge of the dye and the material to be stained. What happens when we take our slide to eosin, which should be used at about 5. The dehydration process starts, but it also acts to differentiate the cytoplasm, or tone the cytoplasm.
Eosin is an acid dye, it has a negative charge, it loves the positively charged cytoplasm, and this affinity is called acidophilia.
I want to tell you just for your information, there are two basic types of eosin. However, when rinsing in water you are kind of diluting your 95 a little bit faster. Now we get back to dehydration. Have you ever noticed how tissue in a histology lab is kind of like rollercoasters? Once you know what one solution does somewhere, it does the same thing wherever it is, it just may serve a different purpose. Here we go to dehydration to remove all the water.
Either you can have a 95 and two s, or three s, depending again whether you want to tone your eosin. Then we go into clearing, and if we recall, clearing is the process here of rendering the cells optically optimal. This little slide right here represents, as far as I know, the only true macroscopic test for recognizing whether your slides have been dehydrated or not.
This is important whether you go straight to an automatic cover slipper or whether you cover slip by hand. Have you ever thrown mercury on the floor, just fun, playing with it, way back in the day? You should not send that to the cover slipper. What you want to see is the middle of the windshield, which I lovingly refer to as the Rain-X effect. The Rain-X effect; just remember that.
How do I know? So I would go through a few pretty obvious ideas, and then we would come up with which they were staining with. Harris is very popular in the U. It continues to oxidize the hematoxylin powder, which we know now is called hematin. Mayers is fairly common, used in Europe; it also oxidizes and needs filtration.
It typically uses a progressive stain. Yes, there is a real definite definition of regressive and progressive, but there are some gray areas that we will look at. Regressive stain; tissue is intentionally over-stained then rinsed in water. This is a strong acid and it will break the covalent bond and it will remove the hematoxylin. In differentiation, rinsing is crucial to remove all the acid. If you hesitate at this point it could cause removal of too much nuclear stain, thereby producing a light stain.
Once we get into this pH environment, our hematoxylin changes. It changes to a red soluble color and the complex has been compromised. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide.
Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume A Rapid Hematoxylin and Eosin Stain. ASCP , J. Laboratory Service, Rodriguez U. Oxford Academic.
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