How to Create a High-Quality H&E Staining Protocol

Good morning, Good afternoon, everyone. Thank you very much for joining. My name is Andrew Lisowski. I'm the technical application manager at Leica Biosystems and today, I would like to present a webinar on how or why quality matters. We'll talk about optimizing H&E efficiency and I'll show you how to create a high-quality protocol that will be bulletproof. 

Let's talk about the objectives. As I said, we will talk about H&E, of course, we'll talk about routine staining and what really does it mean? I will talk about the routine workflow, the steps involved in the workflow, all the steps leading to H&E staining and that is grossing fixation and declassification, processing, microtomy; all those steps might and will affect staining. We'll talk about the staining procedure step by step. I'll talk about the different staining methods and at the end I'll show you some good examples of these staining protocols. Of course we'll close with a good one slide summary. 

H&E or Routine Staining

Let's talk about routine staining. We keep hearing this term routine staining or H&E. Routine staining means something that is relatively fast and relatively inexpensive, that is called routine staining. But before I even talk about routine staining, we need to discuss why we need to stain in the first place. We stain the tissue because the unstained tissue is transparent. You can't really make up any features of the cellular features, so we need to give it a coloration. And so that's number one method around the world. I would say I'm 90% of the slides would go through that method. And of course there is something called advanced staining. The advanced staining, immunohistochemistry or in situ hybridization, those special stains or special staining are ordered when the routine staining will not answer all the questions or when the pathologist is in doubt and he will ask you to do something special. Today, though, we'll only talk about and concentrate on the routine staining. 

Typical Histology Workflow

What you're looking at is a typical workflow through anywhere in the world, you go to Paris, France, you go to Moscow, Russia, or you go to Johannesburg, South Africa; all those steps will be there for the workflow. The instruments might vary, the consumables might be different, but the workflow most likely will be almost identical. What you're looking at really a lot of steps: grossing, fixation, all those steps will take time. And you can see in red staining is towards the end. All those steps preceding this step might and eventually will affect the quality of the stain. When we see something unusual during the staining, we really don't want to diagnose or troubleshoot the staining itself. We need to go back, put that Sherlock Holmes cap on and think what could go wrong and all those steps that would lead to the staining step. 

Let's talk about those steps for a second. So grossing right? Grossing is the step where you make a specimen or a tissue given to or sent to a histology lab, fit a histology cassette. A very common mistake is where people gross or trim the tissue little too thick. We always preach that tissue needs to comfortably fit a cassette which is 4mm deep. If you do four or five and press the lid and force the lid to close, you might have problems with the fixation and later on with processing. So thick issue will not give you the idea of coloration or staining. And if the processing is not correct, that staining will be affected as well. Don't forget that the tissue to volume ratio is very important. We would say it has to be at twenty to one. I've seen ten to one but twenty to one it would be optimal for this perfect fixation. 

How Can Grossing and Fixation Affect Staining?

The fixative will eventually deplete with time and will exhaust its function less. You must remember to put that piece of specimen to a jar that contains the fixative and a 20 to 1 ratio. When using fixative, we need to think about good quality fixative. When I say quality, a known pH, so we all want the fixative to be between 6.8 to 7.2 ideally. You don't have to be a chemist to know its pH neutral. Once the formalin pH, whether you make a mistake because you make it yourself or you purchased from some suspicious source, it's really not a good quality that pH goes down toward the acidic, might be five or six. Once that happens, you might see something called a formalin pigment within the slides, and you won't see it until you stain the tissue.

The formalin pigment looks quite interesting, quite nice. Some new people on the lab might be confused and might think this is maybe red blood cells nicely stained with something else that is just within this tissue, but that is the formalin pigment or the acid formaldehyde hematin that is formed due to the low pH of the formalin. This is a very important thing to remember about using a good quality fixative. The last bullet point here duration, is absolutely crucial to remember. This step is size dependent, so small tissue, little time. Large tissue, more time. A good histology tech will know how much time is needed, but you cannot really rush it. If you rush it, the fixation will be not complete. You will not be able to microtome or section such tissue. And even if you do, the coloration of your stain will be different from the properly fixed tissue. So again, size and time goes together. You have to remember about that.

How Can Decalcification and Processing Affect Staining?

Next, is the decalcification. This is the extra step that is employed when you are working with the tissues that contain ossified cells, right? Bony tissue, anything bone would be decalcified. Decalcification means removing calcium or calcium salts. Hydroxyapatite are the calcium salts that need to be removed in order to work with tissue like that. You need to remove it for the before you process them. The choice of the acids that does the decalcification would be dependent on the size of the tissue. If you're dealing with small biopsy of bone marrow, of course you don't want to use strong acid. You would use weak acid. The weaker, the better. You will have to control time if you are working with the large piece of tissue which is very hard. You don't want to spend days or weeks using weak acids. At that point you will reach out and use a strong base acid decalcifier. Very important: not all acids are the same. There are strong and weak acids and you have to be aware which is what. If you overdo it or forget to remove the tissue from the jar containing strong acid, you will lose the morphology. On the other side, if you undo it, inadequate time, you will not be able to section because those calcium salts themselves will prevent you from creating a perfect section.

In processing, the appropriate scheduler protocol needs to be used, similarly to the decalcification or fixation. Under processing would be something that gives the histologist a big headache because you will most likely not be able to even section such tissue. Using optimal reagents, hopefully reagent alcohols. Reagent alcohols, by the way, are those alcohols that's not ethanol. Ethanol is very popular for some of us in histology labs. Reagent alcohols are mixtures of isopropanol, methanol and ethanol, and they are relatively safer, much less expensive than ethanol and used in gradually increased concentrations. This is the key: increased concentrations. We see a lot of people using alcohols on the tissue processor that would start from 70% and they would jump to 100% concentration. While that might work, this is not really optimal. You want to really increase the concentration gradually to remove that water from the tissue very gently and slowly. And only by doing it in several steps you can be really successful. So gradually increased concentrations means you start with 70%, you move to 80%, 85%, 90%, another 90% and then three times at 100%. At that point you do remove all the water and you can proceed with the solvent. And talking about the stain, some people I know use isopropanol, so isopropanol FYI might change the coloration of hematoxylin. So while the hematoxylin ideally would be this beautiful blue or purplish blue. People that use isopropyl might say, “my hemotoxin looks kind of grayish.” Nothing to do with the staining protocol. It would have to do with the reagent on the tissue processor.

Things to remember is to increase time when you decide to use xylene substitutes. There are solvents that are safer than xylene, so limonene-like products, anything like that would need to increase time and rotate them more often because those xylene substitutes are not as water forgiving as xylene. So you have to remember that. If you don't do that again, the alcohol is not completely replaced and then you'll have a problem cutting the tissue and then staining.

How Can Microtomy Affect Staining?

One other thing. The thing that would lead to the staining quality is of course, microtomy or sectioning. A common mistake is people will tell us, “Hey Andrew, we have you know different results.” A pathologist is complaining some sections are darker, the other sections lighter. What we would find out is just a simple mistake and people not even aware of it. You have several technicians in the lab, maybe an older microtome. It's very easy if you think about it. The average thickness of the sections cut in the histology around the world is about four to five microns. If someone makes a mistake, let's say, and the lab agrees or pathologist wants this tissue to be sectioned four microns, but someone somehow cuts it at five microns. That section will be darker because 20% one micron would constitute about 20% of the tissue because it's thicker. That 20% would absorb 20% more dyes, hematoxylin and eosin, so the tissue will look different.

Our first thing is to really check the settings on the microtomes and do the preventive maintenance if this is an older microtome and the microtome is not keeping the preset thickness. Using the slides is a big one. There are two types of slides. There are what we would call premium slides and economy slides. Economy slides are not coated with anything. Premium slides would be coated with something. A premium slide, in my mind, would be a slide coated with polymers. You can create coated slides in your own lab using a variety of different proteins and if you've been around on the lab for a while, you know people can use albumin or a gelatin, which is the hydrolyzed collagen. Some people with casein. Anyway, those proteins are okay. They would hold the tissue because that's the goal, right? To hold the tissue, to protect the tissue. While the proteins will hold the tissue, they will always create a background.

The background comes from the carboxyl groups and the protein, and there's nothing you can do about it. You must deal with it. People sometimes in there's too much background because someone put too much albumin or gelatin to into the water bath. They think maybe there is too much time; immersion time and hematoxylin. While this might be the case, most likely you need to think about the slides. Are the slides that you make yourself left with just too much up proteins on the surface? Those proteins will cause that background.

Premium coated slides, as I mentioned, these are polymer coated. These are not proteins anymore. Polymer coated slides would not have carboxyl groups that would bind non specifically to hematoxylin. If you have this option, think about using premium slides Premium, i.e. silane or polymer coated slides that would not give you the background at all.

What about additives? While protein-based products are ok and you can use them to bind your tissue to the glass side, they will give you that background I was just talking about because those additives are protein-based. Protein-based additives again, blue background, mostly unwanted. Some people don't mind, but really if you wanted to create a beautiful slide, you want to avoid those protein additives.

And of course, there are a lot of things to talk about when you do a sectioning; floating those beautiful ribbons on the water bath, all about the temperature, and people ask us what is the correct temperature? Which section do I need to pick up? And we always say, well, this smart thing is to pick the section that looks good to you. There is no folding, no bubbles. Nothing like that. And never pick up the sections that are coming off the very first two sections because they tend to be thicker. Pick up the good looking section that will be in the middle of that ribbon.

I think it's time to talk about the staining procedure itself. What you're looking at is a simple diagram I created. It looks like bunch of different reagents from left to right as the staining pathway goes. The staining procedures, as you see, are 20 or so steps depending on the lab. They could be as little as 15. I've seen up to 28, 30 steps. Anyway, the idea is the same. You must run your slides with variety of steps. The typical sustaining protocol would last from 25 to maybe 45 minutes, so relatively fast compared to advanced staining. The number and immersion times would vary depending on who you talk to. It depends on the lab. You remember when you are using while you utilizing the automatic stainer. Please control the water pressure. You can see in gray, water rest stations. These are either if you do it by hand, you just put your water on yourself, there's no water pressure whatsoever. But if you're using an automatic stainer, the water pressure control is very important. You don't want to have the pressure too high or too low and we'll talk about it just in a second. If possible, if you do use that automated stainer, please do employ agitation for uniform stain. One more thing that I forgot to mention toward the right, you can see X for solvent. I decided to use X as a solvent for xylene. Xylene is still the most popular solvent around the world and A for alcohol.

Deparaffinization or Dewaxing

Let's talk about all of those steps individually. The first couple of steps, as you can see beautiful flashing steps. These are the dewaxing or deparaffinization steps. And these are typically done by xylene or xylene substitutes, and those steps are there to remove the paraffin that is adhered to the glass while you are placing the section straight from the water bath. If you do not do this correctly, if you try to rush and let's say you do 30 seconds and then 30 seconds you will experience an unstained area of the tissue, and you might wonder if there's something to do with the hematoxylin immersion times. Most likely this is still paraffin or wax attached or covering the tissue and it's preventing the dyes to create a chemical reaction. The unstained portions are due to the paraffin or wax covering the tissue. The time has to be adequate, right? So let's say a minute minimum in my mind maybe two. Some people would just to make sure there's no paraffin left, they would put a third station so instead of two flashing, you would see three flashing steps.

Hydration

Next you will see something called hydration steps, so three steps. These are the A for alcohol and this is where you introduce your slides to alcohol. Those alcohols start with 100% concentration and the last one to the right, the third one most likely will be 95% or maybe a 90% concentration. That's because the very first dye or very first step, staining the slide would be hematoxylin. Hematoxylin is water based, so you don't want to have 100% alcohol only. You want to add a little bit of water to it, hence 100%, 100% and maybe a 90%. Then a quick water rinse with the right pressure to remove all the alcohol from the slides before the stain. You don't want to rush it again. You don't want to have the times limited to 30 seconds or maybe even two stations, because at that point, not only are you introducing your slides into the aqueous environment, you want to remove xylene that were preceding. You want to remove xylene at that point completely and you are introducing the slides to the aqueous environment coming up.

Primary Staining

After a brief water rinse to remove all the alcohols, we are at the primary staining. Primary staining means that's the first stain or first dye in line. Hematoxylin , which is a blue dye, comes from the tree that grows in Central America. It is a very old dye that histology people used for at least 150 years. As a side note, I always like to say that hemotoxin was known to Aztecs and Mayans in the beginning of the 16th century, and it was used as a dye.

Going back to the real world, hematoxylin stains the nucleus of the cell. Hematoxylin is positively charged and will stain anything that is negatively charged within this tissue or cells. And what are negatively charged? Nucleic acids; DNA, RNA. And where are they located? They are located in the nucleus, so you can say in general hematoxylin stained the nucleolus because that's where the majority of DNA, RNA occupies. All those beautiful blue dots after you're done staining, that is hematoxylin. That is attached to the phosphorus groups of the DNA are most likely within the cell within the nuclei.

Troubleshooting. A few things people will often ask us do I filter my hematoxylin or do I not? The rule of thumb is you do filter regressive hematoxylin and you don't need to filter progressive hematoxylin. People say “okay, fine, which one is the regressive the best?” The best representative, or the regressive hematoxylin is Harris hematoxylin. That's the one that people would have to filter. You filter out something called aluminum ammonium salts. These salts are added to the solution, to the formulation, as a source of aluminum. All the Hematoxylin currently, most of them in the world, are aluminum-based. Some of them are iron, but most of them are aluminum. That salt is needed for the hematoxylin to bind to aluminum. Aluminum is needed as a mordant to attach to the DNA or RNA nucleic acids. You need aluminum. Aluminum could be as supply as a salt and Harris hematoxylin or regressive hematoxylin will have a lot of it. This is normal occurrence. This is expected that the excess of the salts every day will go to the bottom of the vessel, whether this is a cuvette or bottle, so you would have to filter it.

On the other hand, progressive hematoxylins like Gill’s, Mayer, and all the other hematoxylins used in the kits, used by different manufacturers. They are progressive. There are five times less the aluminum ammonium salts, so there's nothing, there's no excess of it, so nothing precipitates. Nothing goes down, so there's no need to filter anything out. Another thing that people think, “okay, what's this gold sheen? I left the hemotoxin over night and there's this gold sheen that forms on the surface of my Harris. This is the effect of oxidation of hematoxylin to hematin. And that's only true for Harris or regressive hematoxylin. You need to remove it. People would use a piece of paper, tissue you have to like quickly skim it from the top. Just remove it from the top, which takes a few seconds.

Now, with the progressive hematoxylins, you don’t need to do that because what all the manufacturers are doing, they're adding polyethylene glycol which is anti-freeze. It's all close to the antifreeze. Almost the same formula. It goes to the top because it's lighter than everything in that formulation. And it protects the hematoxylin from being oxidized. So progressive hematoxylin can't really oxidize or utilize the oxygen from the air, and so there's no effect. There's nothing that sits on the top of the hematoxylin.

If you see something like that in the progressive hematoxylin, that means something went wrong with the manufacturing. You probably shouldn't be using this hematoxylin unless you do filter it, but in general you shouldn't filter and you shouldn't be getting the gold sheen from the progressive stains.

Differentiation

The next step would be after quick water rinse to remove the excess hematoxylin. This is a pivotal step in my mind, the differentiator. People would use either strong or weak acid and this is one of the biggest mistakes with many people, non chemists. They run out of the acid. They would just grab acid from the shelf or from the cabinet. You must know if you're using regressive hematoxylin, when you use Harris hematoxylin. Harris hematoxylin is very, very powerful, right? You filter it, you stain it for a few minutes. People do five or seven minutes. It tends to be very dark, right? You need a very strong acid here and that step differentiation, you need to differentiate this hematoxylin you need to remove all the excess hematoxylin. For that you need always strong acid. Most likely would be hydrochloric acid, about 10% or 14% dissolved in water. For regressive hematoxylin, you need strong acid.

Now if you're using progressive hematoxylin, progressive stain, you don't use strong acid. You use the weakest possible asset you have in the lab. Most likely would be maleic acid, tartaric acid, acidic acid. Anything is very weak because you don't remove the excess of hematoxylin, you are only removing non-specific staining from already stained protein. Remember when I was talking about the slides that people would do would call their own slides with the proteins and those proteins will give you this background because they do have those carboxylic groups that lack hematoxylin. At that step, if you use that slide, you can use weak acid to clean the background to cut off the bond between carboxylic group and the hematoxylin. You can beautify the slide. The times are usually 30 seconds, maximum minute and a half. You don't need much longer. Weak acid for the progress stain.

Troubleshooting. You can imagine people would switch. They would use hematoxylin, let's say Mayer hematoxylin or Gill’s, or something that is proprietary for Leica or other competitors that we have out there. These are hematoxylin that are part of a kit, they are progressive. If you use a strong acid, because you made a mistake, hydrochloric-based acid or differentiator, you will remove most likely all of the hematoxylin or most of it and the staining will look very pale and weak. People would call you or call us and say “hey, there's something wrong with your hematoxylin.” People will blame hematoxylin or maybe timing; the protocol is wrong. Well, you must put your Sherlock Holmes cap on and investigate. Ask people, and then once we learn they used the wrong acid, everything's clear. Acid was too strong and simply removed all the hematoxylin staining. It is very important to know which hematoxylin you use, which type of hematoxylin it is, in order to use the right differentiator.

Differentiation by Staining Method

It is important to know which hematoxylin you use. once you know which hematoxylin it is, then it will tell you which acid to use. My last two-minutes about the hematoxylin, regressive hematoxylin, strong acid. Once this is strong acid, you don't need much time and you can see my stopwatch; seconds. People have that for one dip, three dips, five, ten seconds. Don't forget it's a very strong acid. It removes excess of hematoxylin and it brings it to what it needs to look like. This is a regressive method. The other type is a progressive hematoxylin and here you use weak acid. There's a lot of different weak acids you can use out there. You can see on here and my stopwatch was no longer, so it's longer than three seconds, most likely to be, let's say, a minute. This is something very important for you to remember. This is one of the biggest mistakes people will do. They would simply not know simply which type of hematoxylin they use, or they do, but they still use the wrong acid.

Bluing

We do have your hematoxylin, we use the correct acid, we rinse the acid and we are into the next step called bluing. The bluing step is simply converting beautiful purple hematoxylin into a beautiful blue hematoxylin, and you might say, “wait a second, didn't you say hematoxylin is blue?” Well, if you make an experiment and stop your staining just to see how the tissue looks, how the cell looks, at the hematoxylin stage or step, it's purple because hematoxylin is purple. We don't want to have hematoxylin purple. We want to have it blue.

You might say, “what's wrong with purple? Purple would be too close to the second dye which is coming up to the right. Eosin gives you different shades of red. Sometimes it's kind of reddish, purplish. It would be dangerously, or could be dangerously too close to purple. To make a bigger difference, or differentiate between those two dyes, we would turn hematoxylin from purplish or purple into beautiful blue. All you need to do is a right bluing solution for 60 seconds. One of the cheapest reagents on the market are fairly easy to make it yourself. You can use your tap water. We'll talk about it in seconds or any commercial product. All you need is a solution that has a pH of eight. Why? Because at that pH and we don't know how the science works, but we know that at pH of eight that conversion from purple to blue happens. You don't need much time, 30 seconds to 60 seconds maximum, this conversion happens.

When we evaluate sites and we are asked to help the customer because they say “hey, my slides are too pinkish or nuclei look almost eosin.” We all know right away they are probably not using the bluing solution correctly. Maybe the bluing solution is not what it needs to be, or maybe simply the pH is not where it should be. Our people for that step would use ammonia solutions, they use different lithium and magnesium salt solutions and tap water. Tap water is good, right? Because tap water is slightly alkaline so it tends to be toward eight, so it's 7.6, maybe 7.8. However, tap water’s pH might change. It depends on the village or town. Imagine a tap water in Chicago one day, Chicago officials will add maybe chlorine to kill bacteria and the pH goes down to 7 or less.

If you live in a small town, village people, officials, engineers for the village decide they need to add something else to the water solution, even if it's temporary. That would affect your staining, if your stainer is connected to the tap water source. People keep forgetting about it, “hey, I have a weak staining today and we've seen that more than once. We would send our engineers, our scientists and we would simply test the pH of water and “oh my God, pH is six. No wonder. This is acidic water actually. And the acidic water causes very weak staining.” There's almost a very pale, very purplish staining, nothing to do with the products that you purchased from whoever. It has to do with the tap water. We would say “please if you can, and again bluing solution is very inexpensive, you can purchase it most likely at the 20x concentration, so one part to 19 parts of water, very inexpensive. Do that instead of relying on the pH of that water, because that might fluctuate and cause problems with the stain.

Water Wash

Let's go to the next step, which is water rinse. Usually I don't have water rinse flashing up until now. This is the first time that you can see where the rinse flashes. Why would I do that? Because water rinse must be long enough to completely eliminate or remove the bluing water. As I mentioned more than once, this is alkaline solution pH of eight. We don't want any of that to get to the eosin. This step, I'm trying to activate my beautiful arrow. This is just to see the bluing. If you do it for too short; 20 seconds 30 seconds, there is a little bit of bluing solution we'll get via carryover into eosin and the eosin will look pale. People call us, “hey, I have eosin that really doesn't work that well. What can be done?” They will look to increase the time. That doesn't work because bluing solution is still getting into it. The alkaline solution changes the pH of eosin and making it very pale. Our flushing water rinse a minute, maybe even two minutes if you have time. OK, so that's an important step.

Neutralization

The alcohol that goes in front of the eosin, and this is assuming this is an alcoholic eosin. We know in some parts of the world, customers prefer aqueous eosin, then you would not have this step. This is to prepare the slide to enter the alcoholic environment, which is the eosin right, for most of us. So never mind if you using aqueous eosin you would not have that step whatsoever. If you do, that step should contain 80 to 95% alcohol, because that's what eosins are. Eosins on average contain 80 to 95% of alcohol. This is how you want to prepare your slides to enter this step with the eosin.

If for some reason you are purchasing eosin that is, let's say, 75% alcohol, then you try to match that concentration and that alcohol step should contain the same amount of alcohol as eosin. On average around the world, if you talking about the alcoholic eosin it is about 90 to 95.

Secondary Staining or Counterstaining

We stain with the eosin. We are in eosin actually. So few things about the using synthetic dye, nothing interesting like hematoxylin that I love to talk about it. I wish I could go back to hematoxylin again and talked about the tree that grows in Mexico and Yucatan Peninsula. Eosin is unfortunately a synthetic dye created about 100 years ago by German scientists. It does produce three beautiful colors. A well formulated and done eosin will give you three different colors, right. So that's very important.

Troubles with the eosin might come from several different things, right? The immersion times. The poor eosin or some suspicious sources that will not give you three colors, but only two. The bluing solution not being washed out completely, and it's getting into eosin, changing the pH, raising the pH. Eosin will not last as long as other solutions. It exhausts a little faster unless you really monitor the cleanliness, or the quality of it. pH should be monitored. Check the pH of your starting point. If your eosin is let's say 4.3, make sure that it stays at 4.3 as long as possible, right? So carry over the alkaline tap water will cause the eosin pH to rise and at that point you have to replace the eosin.

Dehydration

Removing eosin, dehydration. You dehydrate or remove the water from the tissue sections and you have two or maybe three alcohols. Depends how much time do you have. You can really play with that very step and people don't know that very well. And they would say, “hey, I'm trying to make this eosin a little lighter or a little darker, I have problems.” It's very tricky with eosin right because there's so many different things that can happen to it. You can play with that step, the very first alcohol, to play with the intensity of the eosin. If you use 100% alcohol here, you will retain the same intensity and coloration as the original eosin.

If you want to differentiate it a little bit and some people called that step dehydration, but differentiating eosin you would use 95% or even 90% of alcohol. Why? Eosin as a chemical has high affinity to water, it prefers water over alcohol. If you move the stained slides over from eosin to alcohol that contains 5% or 10% water, that eosin will immediately move from already stained tissues to water making it lighter. You can really play with the concentration of alcohol and with time. If I put 100% alcohol here, I will retain the coloration and intensity eosin as it was. If I use 95% or 90%, I can make it lighter. People can play with the eosin intensity.

Clearing

Let's proceed with the last step, which is xylene. Clearing, this is where you want to remove all the alcohols from the tissue sections. You would use a solvent. Again, if you're using solvent substitutes like xylene substitutes, limonene products, anything like that. You have to increase the time by about 50% or so, and most likely you will be replacing or rotating them more often as again they not as water forgiving as xylene is. I already mentioned it, immersion times, you can see on the two steps in many different levels just to make sure you remove all the alcohol and maybe water and the alcohol that is carried over from eosin completely removed. You would use three clearing stations.

Examples of Staining Protocols

A couple of examples. This is the example of the regressive staining protocols so you can see regressive hematoxylin highlighted in yellow. If that would be Harris, most likely would be 5 minutes and this is where you would use strong differentiator and again not much time needed for that. Seconds, literally three, maybe five, ten seconds, this is all you. On the other hand, if you want to look at the progressive protocol, same thing progressive hematoxylin would be precise time because you will not be removing anything. Let's say three minutes or two minutes forty-five seconds, and then weak differentiator which would be based on a weak acid. These are examples of the good standing protocols. You can see step number one and two and the last two steps only two. But if you're not making 1,000,000 slides a day and you do have time, I recommend to extend those first and last steps, add another step to make sure you have this beautiful staining. You don't have any wax or paraffin left over on the slide and you clear all the alcohols when you are done.

H&E Staining Steps Summary

Let's summarize it. I did what I could in 40 minutes. It's just a topic we could basically talk about for a day, but very quickly dewaxing is the paraffin removal from the section that's addressed on the slide. This is where xylene, xylene substitute are brought in. Hydration is the removal of that solvent and introducing to the aqueous environment. That's why the hydration last step would be maybe 90 or 90% alcohol. The hematoxylin stains, hematoxylin positively-stained, positively-charged would bind to negatively charged molecules, which are nucleic acids. Hence you see those on the right beautiful nuclei stained blue.

The differentiating step is where you use acid. Don't forget that you need to know which hematoxylin you are using. If it's a regressive, you need a strong acid. If you use progressive hematoxylin, you would use very weak acid, the weaker the better. Bluing step, not much science to it. You would convert the purple hematoxylin into beautiful blue and this is done to avoid confusion or avoid hemotoxin being too close to eosin in terms of coloration. And a side note, please do if you can avoid tap water as this is unpredictable. That tap water can change one day you don't even know that you will not notify you that they do this. You will blame someone that the staining looks not what it should be. Eosin staining. Eosin negatively charged would bind to positively charged proteins in the cytoplasm and a good eosin use and would give you three different colors. Three different hues of red. Dehydration. You remove the water from the previous steps. Or maybe if you're using alcohol that is 95%, to play with the intensity of eosin. And a clearing last step is to remove everything and prepare the slides to enter the environment of the mounting media. If you use xylene substitutes in your multimedia as your multimedia, that last few steps should be of based on a xylene substitute. If this is a xylene mounting media, you would use xylene in your automated or manual method. Thank you very much for joining and hopefully that was helpful and I'll talk to you next time.