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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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Biology Powerpoints

We now have a new page all about biology. http://histologistics.com/biology-powerpoints-by-ben-w-snyder/ To start this new page we have been given biology Powerpoint presentations from Benjamin W Snyder.  He created them from teaching a variety of college biology courses at many colleges over the past 40 years.  All of the Powerpoint presentations he has to date will eventually be put on this website to continue his teachings through others.  You may download and use these as part of or entire class materials.  If you have any question about anything related to these biology presentations, you may comment here.  Ben Snyder often looks at this site and may be able to answer your questions.  Here are 22 power points, also converted into PDF format, of one basic biology course.   The titles of these power points include:

  1. Evolution
  2. Chemistry of Life 1
  3. Chemistry of Life 2
  4. The cell 1
  5. The cell 2
  6. Body Organization and Homeostasis
  7. Neuron
  8. The nervous system
  9. Drugs and the Mind
  10. The Endocrine System
  11. The Adrenal Cortex
  12. Endocrine Adrenal Medulla
  13. Contraception
  14. Female Reproduction
  15. Male Reproduction
  16. HIV & AIDS
  17. Development
  18. Sexual Differentiation
  19. Chromosomes and Cell Division
  20. Principles of Inheritance
  21. DNA and Biotechnology 1
  22. DNA and Biotechnology 2
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Identification of Fungus

Mycology, the study of fungus is not something I know too much about especially when it comes to histopathology.  I do know that fungus is not something I want propagating in my organs or tissues in serious quantity.  Certain species can wreak havoc on the internal and external systems of the animal bodies.  The identification of these fungi in tissues is visualized by staining with GMS, PAS and Gridley’s.  The stain fungi are then analyzed under the microscope by a pathologist who determines by size, shape and surrounding tissue changes, what the type of fungus is.  This is a very exact science that takes years of practice and studying to determine a possible diagnosis.  Some histologists may be able to guess at the type of fungus on a slide but only guess.  For a histologist must rely on the shape and color of a fungus determined by the stain.  I do this on a consistent basis, trying to guess what kind of affliction the tissue reveals after staining.  For fungus identification, I have been relying on a website called doctorfungus.  Here I can analyze my slides against a good knowledge base and slides to match.  Thus I feel like sharing this resource with you might help.

http://www.doctorfungus.org/thelabor/histopat.php

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Oil Red O Stain Protocol

This is one of the stains done on frozen tissues for lipids or fat globules.

Safety equipment: Work under a hood with lab coat, gloves, and glasses.
Use on frozen sections only

1. Hydrate slides in running water———————– 10 minutes
2. Stain in working Oil Red O ————————— 10 minutes
3. Rinse in running water ——————————- 2 minutes
4. Rinse in 60% isopropanol —————————– 5 minutes
5. Rinse in running water ——————————- 2 minutes
6. Counterstain with Harris Hematoxylin —————– 20 seconds
7. Blue in running water ——————————– 5 minutes
8. Coverslip with glycerin jelly or aqueous mounting media

Results:
Fat Globules – Orange Red
Nuclei – Blue

Notes:
• For tissues that do not stay on slides, do not rinse slides in water prior to staining.
• Dextrin must be made for new working solution.
• Dextrin is only good for 1 month.

 

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