A genetic disease developing early in childhood, cystic fibrosis (CF) is characterised by the build-up of thick mucus, bacterial infections and structural damage in the lungs. While some theories suggest an initial infection triggers the onset of other symptoms, a recent study monitoring young CF patients found that the accumulation of mucus in the lungs was the first major sign of the disease. Examining fluids washed through sections of their lungs, a technique known as bronchoalveolar lavage, consistently revealed more mucins, the key proteins in mucus, and more solid mucus flakes compared to healthy children, even when no other symptoms were visible. Their mucus flakes (pictured, left) also had a different appearance under the microscope, denser and more granular than those from healthy lungs (right). With mucus so central to the early stages of CF, the researchers suggest that developing safe mucus-thinning drugs may help slow disease progression in young patients.
Written by Emmanuelle Briolat
Image from the Camille Ehre Lab, UNC School of Medicine
Marsico Lung Institute, University of North Carolina at Chapel Hill, NC, USA
Image copyright held by the original authors
Research published in Science Translational Medicine, April 2019
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Quiz Review #3: Mayer mucicarmine, alcian blue and muller mowry colloidial iron
Today’s flavor is: Mucin! that’s a horrible statement to start off with and i’m already hating this. we’ll start withMayer mucicarmine, let’s go.
I’m gonna just begin by saying that I’ve never done this stain and have never seen it done, BUT reading this procedure is making me dread the day where I’m asked to do it. But let’s start at the beginning.
Mucin or mucosubstance is a secretion found in many epithelial and connective tissues. It is generally PAS positive, metachromatic and basophilic. Mucins are often stained with alcian blue and colloidial iron methods, especially if they contain acid groups. They are primary used to provide lubrication and protection from friction, but some membrane bound mucins are also used to control cell adhesion and proliferation. Mucins can belong to several subclasses of carbohydrates:
Polysaccharides
Composed entirely of carbohydrates
Glycoproteins
proteins that have covalently bound oligosaccharide chains
Proteoglycans
long polysaccharide chains covalently bound to small core protein
Now why do we care about finding mucins in tissue? Several reasons:
Some intestinal carcinomas and certain inflammatory diseases will cause the surrounding epithelial cells to secrete large amounts of mucin; it can be imagined as a distress signal in this context
it can also be used to pinpoint the site of a primary tumor
it can help distinguish undifferentiated mucin negative squamous cell carcinoma from mucin positive adenocarcinoma
certain collagen disease are signaled by having too much or too little of certain acid mucosubstances
used as a diagnostic tool for Barrett’s esophagus, in which long term damage to the esophagus from acid reflux gives rise to abnormal cells just above the esophageal sphincter which may in turn become esophageal adenocarcinoma.
mucicarmine stains are a very handy test to detect Cryptococcus neoformans, the causative agent of Cryptococcosis (an opportunistic fungal disease found mainly in immune compromised patients). C. neoformans has a fungal capsule that contains large amounts of glycoprotein.
Fixation:
BEST- Carnoys or formol-calcium (ie alcoholic fixatives)
OKAY- aqueous fixatives such as formalin
AVOID-gluteraldehyde (will lessen stain intensity of acid mucosubstances)
Warnings and concerns:
The mucicarmine stain is fairly dangerous and is one of a handful of stains that should be performed entirely under a fume hood if at all possible. The official Carson version uses a boiling water bath; the technique my lab uses does not bother with this. The hazards for each component of the stain are as follows:
Mucicarmine stain (overall)
flammable
skin/respiratory irritant
Weigert’s iron hematoxylin
corrosive and flammable
inhalation and skin irritant
Mentanil yellow solution
flammable (at high temps; tho aren’t most things?)
inhalation hazard and skin irritant
How it works:
The aluminum in the mucicarmine stock solution act as a mordant and form a chelate complex in which carmine dye is the ligand. This complex has a net positive charge and is attracted to the anionic acid groups of the mucins present in the tissues. This forms a dye lake. Nuclei are stained by iron hematoxylin and mentanil yellow is used as a counterstain to provide contrast.
Control:
Colon, small intestine and appendix are all acceptable controls but try to get them as fresh as possible. Mucosubstances degrade very quickly after death and autolysis makes it difficult or impossible to find goblet cells that contain mucin.
General procedure:
Stain in weigerts hematoxylin, 7 minutes
wash in running water for 10 mins. Check under scope. you want to see black nuclei but not much else.
stain in working mucicarmine soln for 1 hour. Check under the scope. If at the end of this hour you find that the slides have come out dull, you can incubate them for additional time in the oven until they are bright enough. you want to be able to see red mucin and black nuceli at this step.
rinse in two changes of deionized water
counterstain with mentanil yellow for 30-60 seconds; if you’re nervous, dip a few times and check under the microscope.
Expected results:
Mucin and cryptococcus will be deep red to rose, nuclei will be black and other tissue elements will be yellow.
possible Issues and their solutions:
Weak or no Mucin stainin-check your mucicarmine stain. You probably forgot to add alum, so you didn’t get a dye-lake complex, hence no staining. More time in mucicarmine could help. Also keep in mind that stock solution must be kept refrigerated and won’t last for more than a day at room tempureature. It’s also possible that your mucicarmine is fine, but you overstained with either hematoxylin or mentanil yellow; in the case of the latter everything will look kind of coppery orange.
weak mentanil yellow-mentanil yellow is alcohol soluble, so run it down fast (10 dips per graded alcohol max).
Big disclaimer: Carson basically says that mucicarmine is a pain in the butt and that alcian blue and colloidal iron basically do the same things without being as obnoxious. so, onto them.
Alcian Blue 2.5
Stains for acid mucins, aka mucins that react at low pH values. It’s not a good fit for neutral mucins, but it is compatible witht he PAS if you want to stain both acids and neuturals on one slide. The alcian blue 2.5 stains for weakly sulfated mucins; strongly sulfated mucins are better demonstrated with the alcian blue 1.0 stain.
Reagents and their concerns:
3% acetic acid:
corrosive and combustable
respiratory irritant
skin/eye irrirant
always prepare anything using glacial acetic acid under a fume hood with full ppe. It’s not just vinegar folks it’s real dangerous stuff.
always add acid into water, not water into acid.
any excess made should be neutralized with 1M sodium carbonate and flush down the drain with lots of water.
1% alcian blue:
flammable
inhalation hazard and skin irritant
Nuclear fastred
inhalation hazard
skin/eye irritant
How it works:
Alcian blue gets its color from copper. it’s cationic and is attracted to anionic (acid) mucosubstances, forming salt linkages. Once bound the dye is insoluble in both water and alcohol. We counterstain with nuclear fast red for contrast.
Fixatives and control:
Formalin or bouin solution are preferred. Colon, small intestine or appendix make good controls (but again, get em as fresh as possible).
I’ll put the procedure in later, I just realized I didn’t do laundry and I have to go get my scrubs from the basement before i go to sleep FUCK
expected results:
Weakly acidic sulfated mucosubstances, sialomucins and hyaluronic acid will stain dark blue. All other substances will stain pink to red.
Issues:
if it didn’t turn out right then you probably skipped a step. a good trick is to remove reagents from your bench after you use them.
Jesus christ okay last one
Muller-Mowry Colloidal Iron
This bad boy is looking for acid mucins but much like alcian blue it won’t stain strongly acidic ones. It is not as specific as alcian blue, but it does tend to give more intense color. It’s often combined with the PAS to distinguish between neutral and acid mucins.
Reagents and their dangers;
2% potassium ferrocyanide
actually pretty safe, just don’t heat it
2% hydrochloric acid
strong irritant to eyes, skin, respiratory system; real corrosive stuff
fume hood time
full ppe
acid into water the way that you otter
store acids away from bases
12% acetic acid
listen if you dont get that acetic acid is serious business at this point then i can’t help you
Nuclear fast red
scroll up scrub
How it works:
Colloidial iron in the solution is absorbed by the acid mucins in the tissue. The Prussian blue reaction happens and then, you get, some blue. you know how it is.
Controls and slide setup:
this one’s a little different. you need a control slide (non-patient tissue), then a patient test slide and a patient negative slide. the negative slide will recive only Prussian blue reaction, which will demonstrate the presence of ferric ions in the tissue that are NOT bound to acid mucins
Procedure:
hmmmmmmm yeah i;ll fix this tomorrow heckheckheck
Results, hopefully:
Acid Mucopolysaccharides and sialomucins will stain deep blue. Nuclei will stain pink to red and cytoplasm should be a lighter pink.
Issues:
This is a really time sensitive stain. If you go over or under time limits you’re going to be able to tell.
Alright i am real tired i gotta. go to sleep. same time same place next week, I promise I won’t start as late tho i was traveling a lot this weekend.
Lining the inside of our guts, intestinal epithelial cells are exposed to any pathogens we might ingest. A layer of protective mucus, made up of proteins and antibodies, shields them from attack, yet the common food borne bacterium Salmonella enterica can still find a way through. Transmembrane mucins, proteins that lie across the membrane of epithelial cells, create an important barrier, but one of these, MUC1, can be hijacked by Salmonella. Recent research found that cells possessing MUC1 (pictured, with nuclei in blue, MUC1 in green), are much more vulnerable to invasion by Salmonella (in red) than cells without. Salmonella gains entry into epithelial cells thanks to the interaction between MUC1 and one of its own adhesins, surface proteins used by bacteria to attach themselves to potential hosts. Without this protein, named SiiE, Salmonella cannot invade cells through this route, suggesting that treatments targeting SiiE could block this pathway for infection.
Written by Emmanuelle Briolat
Image from work by Xinyue Li and colleagues
Department of Infectious Diseases & Immunology, Utrecht University, Utrecht, The Netherlands
Image originally published under a Creative Commons Licence (BY 4.0)
Research published in PLOS Pathogens, February 2019
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