#mucin

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.