Carmine stain preparation

2.5g Alum potassium sulfate

1.0g Carmine (C-6152 Sigma)

to 500mLs dH₂O

Boil for at least 40 minutes and keep hot.  Filter through Whatman #1 paper and adjust to final volume of 500mLs.  There will be a lot of stain that doesn’t dissolve.  This can be minimized by keeping the solution at boiling while filtering a small amount at a time.

I store this in the refrigerator.  The stain can be re-used several times, when the stain gets depleted it will take longer to get good staining.  I re-filter after each use—but again only through Whatman #1

Whole mount preparation

Collect the fat pads and spread on a clean glass slide.  Allow to air dry a minute or two and then immerse in Formalin.

Fixing and Staining

1. Fix glands in formalin overnight.  Wash in dH₂O for 5 minutes.
2. Stain in Carmine for 1-3 days.  You can see when the stain has penetrated through by checking the back of the fat pads through the slide.  Fatter glands may take longer than 3 days.
3. Dehydrate in 70% EtOH overnight.  Wash in 95% EtOH 60 minutes.  Wash in 100% EtOH 60 minutes.
4. Transfer to Xylenes overnight to clear the gland.  Fatter glands may take a few days to clear.
5. Dip into 100% EtOH to remove xylenes
6. Transfer to glycerol.  Store the glands in glycerol in open coplin jars in the hood until the xylenes are truly gone (a couple of days).

Steps that can be more than one day (i.e. over the weekend):

1. Carmine
2. 70% EtOH dehydration
3. Xylenes