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JoVE – Increasing the productivity of scientific research

I just found this amazing resource for scientific experiments and more.  They have over 2500 videos that teach laboratory fundamentals.   Their video categories include; general, neuroscience, immunology and infection, clinical and translational medicine, bioengineering, applied physics, chemistry, behavior, and environment.  I just watched DNA extraction from paraffin embedded material for Genetic and Epigenetic Analyses.  I can now go try this in my lab.  Wonderful, I hope this helps!

 

http://www.jove.com/

 

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Hematoxylin and Eosin Quality Control

We need to ensure quality of H&E sections, so when reviewing the daily H&E, we are looking for blue nuclei with chromatin and a well stained nuclear membrane. Also we are looking for 3 shades of eosin, light, medium and dark red/pink. Some of this is determined by the kind of eosin used.  If a control is questionable we should run a second control that we are sure of to ensure that the problem, is a staining problem and not a tissue problem. Over decalcified, over heated or poorly processed/fixed tissue can stain poorly even if all solutions in the H&E stain are OK. When doing H&E on frozen sections extra care must be taken if the slides are fixed in Formalin after cutting. Formalin or concentrated formaldehyde can act as a reducing agent. The best way to remedy this problem is to change the hematoxylin frequently. Frequently can be defined by the lab.  Some labs like clinical hospitals, where the volume is high, may determine a frequency based on number of slides, day or weeks.  Clinical labs are usually controlled by the pathologist, therefore whatever the pathologist wants, is what will become the norm. For smaller labs this is not the case.  Generally a smaller lab will inspect the color intensity of the hematoxylin on a weekly basis to determine the right time to change it.  As a rule of thumb, hematoxylin must be filtered before each days use.  This helps to keep the floating precipitate out, removes any extraneous cells and lets it keep longer.  The time in the 95% alcohols after eosin is where the three shades of eosin are differentiated so these times may need to be adjusted for best results.

Rat Liver H&E 20x
Rat Liver H&E 20x

reducing agent (also called a reductant or reducer) is the element or compound in an oxidation-reduction reaction that donates an electron to another species. Because the reducing agent is losing electrons, we say it has been oxidized. (http://en.wikipedia.org/wiki/Reducing_agent)

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Elastic Fiber Pictures

Human skin VVG 20x
Human skin VVG 20x
Human skin VVG 10x
Human skin VVG 10x

Here are the other 2 pictures that were going to be in the previous post, plus 6 more.  The other 6 are also 10x,20x, and 40x of 2 different tissues.  I can not imagine what life would be like without flexibility.

Aorta Verhoeff Van Gieson 10x
Aorta Verhoeff Van Gieson 10x
Aorta Verhoeff Van Gieson 20x
Aorta Verhoeff Van Gieson 20x
Aorta Verhoeff Van Gieson 40x
Aorta Verhoeff Van Gieson 40x
Human Uterus Verhoeff Van Gieson 10x
Human Uterus Verhoeff Van Gieson 10x
Human Uterus Verhoeff Van Gieson 20x
Human Uterus Verhoeff Van Gieson 20x
Human Uterus Verhoeff Van Gieson 40x
Human Uterus Verhoeff Van Gieson 40x
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Verhoeff Van Gieson Stain

Safety equipment: Work under a hood with lab coat, gloves, and 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 running water——————- 5 minutes
  7.  VVG working solution—————————— 60 minutes (re-use)
  8. Wash in running water—————————— 2 minutes
  9. Differentiate sections microscopically in 2% ferric chloride until the elastic fibers are distinct and the background is colorless to light grey.  If the sections are differentiated too far, re-stain. (see notes below)
  10. Rinse in DH2O—————————————- 1 minute
  11. Sodium thiosulfate———————————– 1 minute (re-use)
  12. Wash in running water—————————— 5 minutes
  13. Counterstain in Van Gieson’ s——————— 1 minute (re-use)
  14. Dehydrate in 95% alcohol————————– 1 minute
  15. Dehydrate in 95% alcohol———————— 1 minute
  16. Dehydrate in 100% alcohol———————- 1 minute
  17. Dehydrate in 100% alcohol———————- 2 minutes
  18. Dehydrate in 100% alcohol———————- 2 minutes
  19. Clear in xylene (3)———————————- 2 minutes
  20. Clear in xylene (4)———————————- 5 minutes
  21. Coverslip 

Results:

Elastic Fibers —————————————- Black

Background —————————————— Yellow

Notes:

  1. It is easy to over differentiate.  If the background is completely colorless so that a clear yellow counterstain is obtained, the section may be over differentiated.  It is probably better to err on the side of under differentiating.
  2. Over differentiated sections can be re-stained.
  3. Do not prolong the Van Gieson stain; picric acid could differentiate the stain further.
  4. Be sure to follow the order of adding all reagents when making the Verhoeff’s solution.
  5. To clean the Verhoeff’s stain out of the jar, wash with 2% ferric chloride solution for a few minutes.
  6. For optimal results differentiate the control slide first.

Porcine Aorta VVG 40x