Hematoxylin and Eosin (H&E) staining

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Hematoxylin and Eosin (H&E) staining 

Introduction:

In histology and pathology, hematoxylin and eosin (H&E) staining is the staining method most frequently employed. It is a frequently used method to view the cellular makeup of tissues and to make medical diagnoses. Hematoxylin stains the nuclei of cells blue-purple, while eosin stains the cytoplasm and extracellular matrix pink.

Requirements:

  • Paraffin-embedded tissue sections
  • Hematoxylin solution
  • Eosin solution (1%)
  • Different grades of alcohol (70%, 95%, and absolute)
  • Xylene
  • Acid  alcohol 
  • Ammonia water  
  • Microscope slides
  • Coverslips
  • DPX/Canada balsam

Principle:

First  the  tissue  is  cleared  of  all  wax  and  then  rehydrated  to  facilitate  the  entry  of  dyes. The nuclei, ribosomes, and some cytoplasmic granules in the tissue are acidic and are stained by the basic pigment hematoxylin. Eosin is an acidic dye that stains the tissue's fundamental elements, including collagen and cytoplasmic proteins. These two colors work well together to provide a strong contrast that brings out the cellular features.

Procedure:

  1. Deparaffinize the tissue sections 
    1. Place in hot air oven for 15 minutes.
    2. Then two changes of xylene for 3 minutes each. 
  2. Then rehydrate the section with graded alcohol, going from higher concentration to lower concentration i.e. 100% to 70%.
    1. Transfer to absolute alcohol for 3 minutes.
    2. Place in methylated spirit (95% alcohol) for 2 minutes.
    3. Transfer to 70% alcohol for 2 minutes.
    4.  Wash the slide in running water for 1 minute
  3. Stain the sections with hematoxylin for 5-10 minutes.
  4. Rinse the sections with tap water and differentiate the sections in acid alcohol for 30 seconds.
  6. Give  2-3  dips  in  ammonia  water  solution until the tissues attain a blue colour.  
  7. Rinse the sections with tap water and counterstain with eosin for 1-2 minutes.
  8. Wash in running tap water for 30 seconds.  
  9. Dehydrate the sections with graded alcohol, going from lower concentration to higher concentration i.e. 70% to 100%, (2-3  dips  in  70%,  95% and absolute alcohol).
  10. Clear them in xylene.
  11. Mount the sections with a coverslip using a mounting medium (DPX/Canada balsam).

Results:

Nuclei: Bright blue 
Muscle, keratin: Bright pink  
Collagen and cytoplasm: Pale pink  
Erythrocytes: Orange-red 

QC:

Quality control is essential to ensure accurate staining results. The following measures should be taken:

  • Ensure that the tissue sections are properly deparaffinized.
  • Use fresh staining solutions.
  • Do not over-differentiate the sections in acid alcohol.
  • Do not over-stain the sections with eosin.
  • Ensure that the coverslips are properly mounted and secured.

Interpretation:

Based on the cellular alterations shown in the tissue slices, H&E staining is used to detect a number of disorders. Pathologists can detect aberrant cells or tissue structures as well as the cellular shape, organization, and dispersion of the cells. This staining method is frequently used to diagnose cancer since it can reveal aberrant tissue architecture and cell proliferation.




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