[0:10]Okay, so now turning our attention to the Masson's Trichrome stain, looking at this first slide here, this is a section of tongue. And we can see there's some really nice red skeletal muscle here. We can even see some striations. The nuclei are beautifully demonstrated, they've got that nice uh dark, almost black red color um that is clear. If we go further into the connective tissue, we can see the blue is especially vibrant and again, those nuclei are standing out really nice and dark in contrast to the surrounding collagen fibers. Moving up to the surface of the tongue here within the epithelium, those keratinocytes are staining up also very vibrantly. So just bear in mind that both muscle and keratin within epithelial tissue does stain up quite similar with the Masson's red. So this is a an example of the good level of staining that you'd expect to see. Uh, it appears this has also been post-fixed in the picric acid and that is certainly advantageous to achieve a more complete or even staining or um, sorry, fixation initially within the tissue. And that more even fixation is resulting in a more uh, uniform staining of the of the tissue. So, what can go wrong? Well, one of the things that occasionally goes wrong, and it can happen, I guess, for a number of reasons, and I've never actually sat down to identify what point this occurs. But certainly, it's very clear that there is a problem when we end up with nuclei that look bright red. In fact, they're not turning out as red on the screen, but down the eye pieces, I can see that these nuclei within the connective tissue now, they are at a, well, they they're actually quite red, believe me. And so, that is generally a consequence of the hematoxylin having been exposed to conditions uh that are acidic. Now, the potential sources of that, maybe the slide was not blue after being stained with the hematoxylin, that's one possibility, and I suspect in many cases that that is the the source of the um, of the red staining. Here we've got some nerve bundles, and they are actually showing it quite clearly through there as well.
[2:59]So, um, yeah, most likely the hematoxylin wasn't blue, but I think another potential cause of the red color is if there has been difficulties in differentiating out the Masson's red with using the PMA. Because the PMA is acidic, then potentially that it that longer exposure to more acidic conditions could potentially revert the color of those nuclei back to that more red color. Is it a big issue? Well, not really in terms of the overall quality of the stain.
[3:39]We can see there's excellent staining of the collagen surrounding those nerve bundles there, the the epinurium layers. Uh, the the muscle is also nice and red here. Some of these nuclei through here, probably a few fibroblasts and so on, they're actually not too bad as well. It's just that in those areas where I'm um looking to study the nuclei more clearly, I can see that uh there's a there's a definite lighter color compared to the nuclei that we'd normally expect as was shown in that in that first example. Okay. So I think the general take home message from that, again, it's the same for all the trichromes. Uh, just the one dip in the acid alcohol and one dip only back into the water bath to really ensure that the appropriate level of staining is there to begin with, and then make sure that you do blue it. So blueing is not just for for doing a H and E. It's not just for a PAS with your Mayor's hematoxylin. You also technically blue, although it ends up kind of black, for when you're doing trichromes as well. Now, the other problem that can occur occasionally with doing the trichromes is because you're adding your various stains in in um, in sequential order. Um, you can sometimes accidentally result in more of the methyl blue entering the the areas of muscle or in fact even the keratin.
[5:10]So the overall appearance of the the muscle in this particular slide of tongue is uh more of a kind of a purple color. So if you look back to what we saw earlier in the preceding slides, where the muscle was a nice definite red or even burgundy, but here we've got the the layers um here of well, these areas of skeletal muscle uh quite blue. In fact, if we look on the edge here, this particular fiber seems to have a lot of methyl blue that's that's leeching into it. So, what could have caused that? Well, if we think back to the mechanism of staining with these trichromes, even though there's a difference in the relative molecular weight and porosity, if you leave the stain on for too long, the extra time will actually enable some of that methyl blue to penetrate into the into the muscle and to displace that mixture of red dyes that are that are present. So that's what I believe has occurred on this occasion and it's resulting in this more purple color of the um the the skeletal muscle. So, does this slide have diagnostic value? Well, of course it does. You can still clearly see the collagen really nicely. It certainly doesn't prevent it from having value. And even up here in the keratin layers, we can see that the keratin is quite clearly distinguishable from the collagen. It's just one of those minor technical things uh that can be mastered, and um, because we know that you can do it, it's another area that we look for when when we're marking.
