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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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Chemical Compatibility Guidlines

Chloroform
Wet Chemical chloroform

 

This is how I have started storing our stock of wet and dry chemicals.  Most labs have an abundance of both dry and wet chemicals that must be kept for reagent solutions, media mixes, pH calibration and other.  Depending on the size of the lab, chemicals might be stored inside of specially designed cabinets that are prevent accidental spills.  Sometimes the chemicals will be put on a shelf, countertop, refrigerator or freezer.  This is an issue in most of the clinical, research and academic laboratories I have worked in and may be for many others.  Over the years and out of want to be safe, I have designed a suitable solution for storing both wet and dry chemicals that is safe for everyone to use.

DSC02091

 

Chemicals can be hazardous if they leak, spill or come in contact with other non-compatible chemicals.  Therefore is it the duty of someone who cares to make sure this never happens.  For instance, I take chemicals very seriously, not because my mother was a chemist for so many years that she lost her sense of smell but because I was a victim of a small histology company and its lack of safety procedures.

The chemical storage protocol here has been modified from a hazardous waste guideline.  This guideline provides details of specific chemicals that can and cannot go together as a waste stream.  Therefore I conclude that these chemicals should also be stored in the same way.  There are some obvious conditions I would like to point out anyway.  Flammable cabinets should be used, not only for your sake, but for the firemen who might come to rescue you.  This tells everyone to beware.  This also goes for acid and base cabinets.  I feel these are necessary for the Modern laboratory.  Acids and bases do not go together, obviously. Secondary containers are helpful when space is limited and chemicals must be stored close together.  The container would contain a spill or leak from reaching other non-compatible chemicals.

Chemical incompatability
Chemical incompatibility

 

The chemical chart breaks down what chemical can and cannot be stored together, essentially making safety a top priority.  The PDF of the chart is below and will also be placed in the protocols page.

I hope this helps.

How do you store chemicals in your lab?

Chemical_Compatibility_Chart