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Homemade Aqueous Mounting Media Works Great

The earlier 2 posts about the aqueous mounting media we made has a third update.  3 different labs to date has used our homemade aqueous mounting media with great success.  It has been used flawlessly on fluorescent antibody staining and special staining (oil red O) for histology techniques.

The current alternative to making it is buying it from 1 of many companies for an outrageous amount of money.  I wonder if one manufacturer makes large batches of it, then sells it to the different retailers?  What do you think?

The protocol needs a little tweaking as it’s very thick and I don’t think it needs to be.  There’s also the antifade agent (Either p-Phenylenediamine hydrochloride: 100 mg or n-propyl gallate: 500 mg) that is optional to add.  I did add it to the mix, using p-Phenylenediamine and it turned the liquid brown.  After doing this I and others had doubt about its effectiveness, but it worked and the brown color had no effect on the fluorescence or ORO.  I think next time I make it, I will use less antifade.

I remember making this stuff because it took so long, about 4 hours.  Of course, this was the first time and I was doing 10 other things besides this.

As promised, the recipe for this solution is copied here.  This is found on John A Kiernan’s website http://publish.uwo.ca/~jkiernan/aboutjk.htm, histology books and papers he has written or contributed to. Thank you John for giving us your insight into the world of histology, you have truly made difference to so many people!

Polyvinylpyrrolidone (PVP) medium

 

This is my favorite aqueous mounting medium. The composition can be varied according to each need (Kiernan, 1990). For immunofluorescence, make it up in a buffer and add an anti-fading agent.

 

Polyvinylpyrrolidone (M.W. 10,000): 25 g

Water (or a 0.1 M phosphate or

TRIS buffer, pH 7.4 or 9): 25 ml

Dissolve the PVP by leaving for several hours on a magnetic stirrer. Then add:

Glycerol: 1.0 ml

Thymol: One small crystal

Bottles of PVP medium usually keep for 2 to 3 years at room temperature. Keep it in a dark place if an anti-fading agent has been added. Discard if it looks infected or becomes too viscous.

Anti-fading agent:

Either p-Phenylenediamine hydrochloride: 100 mg

or n-propyl gallate: 500 mg

 

This mounting medium is more runny than glycerol jelly or Apathy’s. It is very easy to apply, and not prone to bubbles. The refractive index is 1.46 (Pearse, 1968), but increases as the water evaporates at the edges of the coverslip until unstained structures are barely visible. If you want a high degree of transparency, wait several days before sealing the edges of the coverslip.

 

 

  1. A. Kiernan

Department of Anatomy & Cell Biology

The University of Western Ontario

London, Canada N6A 5C1

 

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Aqueous Mounting Media, Update

Several labs have and continue to use the newly made aqueous mounting media.  So far it has been used for IF only but does do just as well as the commercially bought products. At this point the only difference that has been observed is the color of the liquid, it’s brown.  This does in no way cause any visual problems when viewing the slides under a fluorescent scope, causing no distortion or color skewing.

Amyloid fluorescent

I am hesitant to say this is good for all aqueous mounting types because it has not been used on special staining like oil red O.

I hope to try this on test slides within the next several weeks and will update again as this progresses.

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Aqueous Mounting Media’s for Fluorescent and Special Stains, Commercial Brutality?

We do a fair amount of special and fluorescent staining that requires aqueous mounting media and not the toluene based glue.  Normally for special stains I make up a glycerin jelly that has phenol and must be kept in the refrigerator.  This was taught to be the standard of aqueous cover slipping media.  It never dries, sticks to everything and smells terrible.

The fluorescent slides need something different, usually a special fluor mount bought at one of the large chemical companies.  This mount allows for very little light distortion while viewing through a con-focal or fluorescent scope.  The amount of light distortion is called refractive index.  Some of these mounting medias that having a low refractive index of 1.47 can be very expensive, $162.00 plus shipping for 10 ml.

Triple antibody using the confocal microscope.
Triple antibody using the con-focal microscope.

10 ml may be enough to cover slip 300 slides if using only 30 um per slide but that adds $.60 to the cost of every slide.  That may not seem like much but when it’s added to the total after processing, embedding, cutting, antibody plus working hours it can push the average researchers budget to the edge.

I thought, there has to be something better than this.  Large companies get most of their protocols from the old histology books and change 1 micro-gram and it becomes proprietary.  Then the price depends on demand and repeat customers (way too high).  This is what I call commercial brutality.

I’m way to enterprising to pay these prices and be happy about it.  Therefore I too will develop a protocol from the books and change one aspect of it and call it my own.  The difference is, I will formulate it, test it, have others test it, then make it available to all the labs I have daily contact with (10) and help them save money.

One way to stop this money merry go round is to limit profits from unbelievable pricing on medical products.  Paying $500.00 for a water bath that lasts 10 years is absurd!  $162.00 for 10 ml of mounting media is too much!

I have made 25 ml the histo-lo-gis-tics version of aqueous mounting media for about $20.00, but could be 100 ml for $40.00.  That’s the cost if you make it for yourself.  It’s a room temp, permanent media that will last for 2-3 years with anti fading agent and refractive index of 1.46.  So far it’s been tested by 2 independent labs in fluorescent microscopy and passed with no distortion.  2 more labs will be testing it this week and mine soon.

After testing is done, I will update the results, good or not good and post the recipe if it’s useful.