Question

3- you have decalcified a bone biopsy in a 5% HCl for classical H&E staining. Explain 3 steps that you should do to make sure that H & E staining will be of best possible qualities.

Answer 1

H & E (Hematoxylin and Eosin) staining demonstrates the morphological details of the nucleus and cytoplasm in a really good manner. Steps to ensure that H & E staining will be of the best possible qualities are many. The depth of coloration depends on the amount of DNA in the nuclei and the length of time the sample is in hematoxylin. Hematoxylin oxidation produces hematein and mordant addition improves the property of hematein to attach to the anionic components of the tissues. Eosin is the common counterstain used to distinguish between cytoplasm and nuclei of the cells. The addition of a little acetic acid sharpens the eosin staining.

Three important steps to ensure a good quality H & E staining are:

1. Proper balance of dyes, hematoxylin and eosin, should be achieved. Overstaining with hematoxylin will give an impression of under-stained eosin and vice versa, which should be avoided. Uneven staining should be avoided.
2. Ensure proper complete dewaxing with clean and fresh dewaxing reagents. The residual paraffin often results in uneven staining or unstained areas.
3. Accurate timing should be followed in each and every step of the staining process.