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AFIP Lee G. Luna Stain Protocols

One of the most useful staining manuals for histology is the Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology edited by Lee G. Luna 1960.  This contains some of the most used procedures used in histology for the past 50 years.  Most of our stains used for Histologistics are modifications of these protocols.  The book is packed with many of the “old” recipes for making stain solutions by hand using dry and wet chemicals.  For example, there are 7 different hematoxylin recipes that are detailed with side notes about color and ripeness.  In the special stains sections are many modifications to detect the same type of tissues.  There are 6 different protocols to stain for general bacteria. http://histologistics.com/histology/f/, 5 different protocols to stain for acid fast bacteria. http://histologistics.com/histology/special-stain-techniques-for-bacteria-fungi-and-inclusion-bodies/.  Because this book is no longer in print and has so much useful information, we have decided to copy as much of the book onto this site as possible.  We have already begun, as you see the links above.  The web has tons of histology information on many different websites but not on just one.  When we search the web for histology information, it would be nice to have one site to go to instead of 10-15 sites, each having a little piece of what we need.  This is a long-term project that we hope, will help us and everyone else with all forms of histology.  If you need any help with any phase of histology, post your questions on this site.  If we do not know the solution, we have the resources to find out.  

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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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High School Histology Internship Update

This is the second week of the high school internship program collaborating with WPI.  Their task is to create 25 perfect slides, 5 different stains from 5 different tissues. Each of the tissues are picked by them and must be grossed, cassetted, processed, embedded, cut, stained and microscopic pictures are taken by them.  They In the first week the interns learned all about safety, grossing, processing and embedding.  This week they have been practicing on the microtome.  They have been given 12 tissues to practice facing, sectioning, laying on the bath, separating sections and section placement on slides.   Tomorrow I will explain and show them how to do H&E staining on some of their cut tissues.  We will discuss the elements of each of their tissues grossly and microscopically so that they can choose their next 4 special stains.  After their tissues are all stained, they will write short descriptions of each tissue to highlight it’s unique composition.  Follow this program with me for the next 6 weeks.

 

 

An Expert Histology Service  

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Mallory PTAH Stain Protocol For Muscle Striations

This is an old stain not used much anymore for diagnosis.  It has been replaced by immunohistochemisty.

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. If sections are formalin fixed, mordant in Zenker fixative containing 5% acetic acid overnight at room temperature.
  8. Rinse in running water ————————— 2 minutes
  9. Place in Gram’s Iodine ————————— 15 minutes

10. Rinse in running water ————————— 1 minute

11. Place in 5% sodium thiosulfate ——————– 3 minutes

12. Wash in running water ————————— 10 minutes

13. Place in 0.25% potassium permanganate ——— 5 minutes

14. Rinse in running water ————————— 1 minute

15. Place in 5% oxalic acid ————————— 1 minute

16. Stain in PTAH at room temp ——————— Overnight

17. Dehydrate in 95% alcohol————————- 30 seconds

18. Dehydrate in 100% alcohol———————– 30 seconds

19. Dehydrate in 100% alcohol———————- 30 seconds

20. Dehydrate in 100% alcohol———————- 30 seconds

21. Clear in xylene (3)——————————- 2 minutes (re-use)

22. Clear in xylene (4)——————————- 5 minutes (re-use)

Results:

Cross-striations, fibrin – Blue

Nuclei – Blue

Collagen – Red-Brown