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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.

 

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Poly-L-Lysine

We recently had a request for the protocol to coat slides with poly-l-lysine.  This is good for tissues that fall off using positively charged slides or chrom-alum in the water bath.  Sometimes tissues just will not stay on even for just H&E staining.  This can be frustrating and bothersome.  Frozen sections and sometimes IHC will give these kinds of problems.  A histologist will typically try different solutions until finding one that works.  The protocol will also be posted in the protocol section for printing.

Poly-L-Lysine

Make your own concentrated solution: 

Poly-L-Lysine – 1gm

DH2O – 100ml 

Or

Buy pre-made Poly-L-Lysine 

1. Dilute stock solution 1ml Poly-L-Lysine to 10ml DH2O

2. Prepare a 50ml working solution in a coplin jar

3. Place plain glass slides (no charge) in the solution for 5 minutes

4. Take slides out and air dry (slides must be completely dry before using)

5. Repeat as necessary

 

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Alizarin Red S Stain Protocol for Calcium

Run a control slide. Control tissue: Bone or tissue with calcium present

Safety equipment: Work under a hood with lab coat, gloves, and safety glasses.

  1.   De-paraffin slides in xylene (1) —————— 2 minutes (re-use)
  2.   De-paraffin slides in xylene (2)——————- 2 minutes (re-use)
  3.   Clear slides in 100% alcohol———————- 2 minutes
  4.   Clear slides in 100% alcohol———————- 2 minutes
  5.   Hydrate slides in 95% alcohol——————– 2 minutes
  6.   Hydrate slides in 50% alcohol——————– 1 minute
  7.   Rinse rapidly in DH2O—————————— 30 seconds
  8.   Cover the sections with alizarin Red S solution (re-use)
  9.   Observe the reaction under a microscope and remove when the red-orange lake forms ( 5 minutes to 15 mintues). The lake should be heavy but not too diffuse.

10.    Shake off excess dye and blot carefully.

11.    Dehydrate in acetone for 10 to 20 seconds (dump)

12.    Dehydrate in acetone-xylene (50:50)———–10 to 20 seconds (dump)

13.    Clear in xylene—————————————- 5 minutes (re-use)

14.    Coverslip

Results:

Calcium Deposits (except oxalate) ———————Orange-Red

This precipitate is birefringent.

 

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