Hematoxylin and Eosin (H&E Stain)

00:00 Let's now look at the basis of hematoxylin and eosin staining.

00:08 Having already stained a slide after preparing it using the technology I briefly went through in the first part of this lecture, let's look at staining.

00:22 Hematoxylin and eosin is the most commonly used stain or dye in histology.

00:28 An example of it is on the right-hand side image.

00:32 It's a common slide that I've used in a previous lecture.

00:38 Hematoxylin is a basic dye.

00:41 It has a net positive charge on its colored portion, whereas eosin is an acid dye or an acidic dye.

00:54 It has a net negative charge on its colored portion, and just briefly on the right-hand side, when you use hematoxylin and eosin, it stains different components different colors.

01:10 Without going to the details yet, hematoxylin produces that bluish stain you see associated with those components in the tissue we'll go through in a moment whereas eosin stains the tissue components a pinky-red color, and that's what you'll commonly see in most of your slides in your class slide box.

01:34 Let's just try to understand what we mean by the term basophilia.

01:41 Basic dyes like hematoxylin are going to react with the anionic components of cells and tissues, that is, those carrying a net negative charge.

01:57 So, when we look at anionic components of cells and tissues, when they react with a basic dye, those components are said to exhibit basophilia, and basophilia or the degree of basophilia actually reacts or depends on the pH used during the staining process.

02:26 Some of the anionic components of cells or tissues that react with the basic dye are the carboxyl groups of proteins, phosphate groups of nucleic acids, and sulfate groups of glycosaminoglycans.

02:44 And I certainly don't want to go into the chemistry of all these components in this lecture.

02:49 It's just to give you the basis for what the staining technique used actually targets in the tissue components.

03:00 If you use a pH of about 10, all those anionic components are going to take up the basic dye.

03:10 If you restrict the pH to between 5 and 7, in other words, it's slightly acidic to neutral, then you're going to only stain those components of the tissue or the cells.

03:26 And if you restrict the pH to less than 4, then you're going to only take up the stain of the sulfate groups of the glycosaminoglycans.

03:41 Acid dyes on the other hand, let's just define what we mean by acidophilia.

03:48 Acid dyes like eosin react with the cationic components of cells and tissues especially ionized amino groups of proteins.

04:03 So acidophilia, when we look at the cationic components of cells and tissues that react with an acid dye such as eosin, then those components are said to exhibit acidophilia.

04:21 In the case of reacting with eosin, we sometimes refer to them as being eosinophilic as well as being acidophilic.

04:33 One problem or 1 issue about acidophilia is the reactions are not as specific as the reactions are with basic dyes such as hematoxylin.

04:45 Let's again look at a few slides.

04:47 You've seen the one on the left and now, there's another one on the right-hand side.

04:53 Very dark blue tinge to that tissue. It happens to be cartilage.

04:58 And what it does is it exhibits basophilia, and it's going to illustrate the number of substances within cells and other external parts of cells that might display basophilia that you'll come across in your histology course and is shown here.

05:24 For instance, the top section you've looked at before.

05:29 All those nuclei stained with hematoxylin because the heterochromatin and also the nucleoli contain ionized phosphate groups in the nucleic acids.

05:48 The endoplasmic reticulum of cells often collectively termed ergastoplasm and also ribosomes contain ionized phosphate groups on the ribosomal RNAs.

06:07 They also exhibit basophilia so they're going to stain a bluish stain with the basic dye of hematoxylin.

06:18 And I'll show you an example of that in a moment because often, it's hard to see the endoplasmic reticulum and even ribosomes in cells where they might be making protein components that might be eosinophilic and totally dominate the cell cytoplasm.

06:34 And lastly, extracellular material such as the matrix of cartilage contains lots of ionized sulfate groups in glycosaminoglycans, and you'll see on the right-hand side an example of the matrix of cartilage here that exhibits basophilia.

06:53 It's reacting with the basic dye or hematoxylin.

06:58 Acidophilia or when we use eosin, it's sometimes referred to as eosinophilia, that staining can exhibit a number of different tissues.

07:14 It can show up different tissues of the body.

07:18 For instance, on the right-hand side of this image, we see smooth muscle cells.

07:25 They're the homogeneous pink-stained block of tissue you see on the right-hand side.

07:30 So eosin or acidophilia is exhibited by those cytoplasmic filaments inside the cell such as the contractile filaments, contractile proteins.

07:44 Also within cells, some of the membraneous components of cells, always difficult to see with the light microscope, they also will exhibit eosinophilia or acidophilia and stain with the dye eosin.

08:00 And on the left-hand side of this image, you can see extracellular fibers in the case of collagen, this is, and again, these ionized amino acid groups exhibit acidophilia and stain this pink stain with eosin.

08:21 Again, cytoplasmic filaments in skeletal muscle fibers exhibit acidophilia.

08:28 And here's an example where you can see both stains used together to distinguish typical cell components, again, like I've mentioned before, you will come across in most of your histological sections.

08:44 On the left-hand side is a section through the stomach.

08:49 You can see a number of different staining features here.

08:54 For instance, on the very surface, you see these invaginations of an epithelium.

09:00 Don't worry about the details. And those invaginations have clear staining mucous cells on the surface.

09:10 That mucus is lost during processing.

09:14 It's lost during the alcohol dehydration of processing that I spoke about before and therefore, there's nothing there to stain so that appears very clearly.

09:29 Down the bottom into the tissue, you see a population of pink-stained cells and a population of bluish-stained cells with a few pale-stained cells in the middle.

09:42 They're cells that are exhibiting acidophilia and basophilia, staining with eosin or hematoxylin.

09:52 Move over to the right-hand image now where you see a portion of that tissue.

09:58 It happens to be a section through the stomach in higher magnification.

10:05 The very dark pink-stained cells exhibit acidophilia or eosinophilia.

10:14 They're eosinophilic, they're reacting with the eosin dye.

10:21 And a lot of the reasons why these cells are staining a bright pink is because they are full of mitochondria, which are eosinophilic or acidophilic components of cells, and those mitochondria are very important in producing hydrochloric acid which is a product of the stomach.

10:43 The more bluish-stained cells down the bottom are exhibiting basophilia.

10:53 The nuclei stain are very dark purple color and here, you see that the cytoplasm, unlike the red-stained cells I just mentioned which are called parietal cells, the very pale blue-stained cells you see towards the base, they're chief cells of the stomach.

11:15 They're synthesizing pepsinogen and that requires an enormous factory of endoplasmic reticulum.

11:25 And if you remember back a few moments ago, I said endoplasmic reticulum is highly basophilic, and you see it clearly here because it's dominating the cytoplasm of the cell.

11:39 Now, one final point about tissue processing is that within that tissue you see on the right-hand side, you can see little strands of whitish areas.

11:52 Normally, that is interstitial space, but it's exaggerated in this slide and it's exaggerated a lot in your H&E stain sections, and that is because when you cut a thin slice of tissue and pass it through dehydration through alcohols, it often gives rise to differential shrinkage of different tissue components, resulting in what we see as false spaces here.

12:23 They don't really occur in the real life of the specimen.

12:27 They're a product of the processing, and you'll come across a number of details in your tissue sections— I won't go through them here— that are a product of the tissue processing, either differential shrinkage or excessive staining and therefore precipitation of the stain, all those processing errors, if you like, are called artifacts.

12:54 They're not a true reflection of the real structure.

12:59 They are a product of tissue processing.