Biñas, Celestina, C.

BSMLS 3-A-IRR 1

H&E Staining

Critical Thinking Questions:

1. What is accomplished by the following procedure in H&E staining process?

A. Dipping in the first xylene
 Deparaffinization - removal of paraffin
 Clearing – displacement of alcohol from the tissue sections with the clearant to assure
miscibility when cover slipping with xylene–toluene or other petroleum-based mounting
media.

B. Dipping in hematoxylin dye

 It stains the nucleus of the cell, specifically the chromatin within the nucleus and the
nuclear membrane. The nucleoplasm of the nucleus should remain unstained this allows
the staining pattern of the chromatin to be seen easily.

C. Dipping in acid alcohol

 Hydration – the introduction of water into the tissue section. This is done by passing the
slides slowly through a series of decreasing concentrations of alcohol.
 Dehydration – the removal of water from the tissue section.

D. Dipping in ammonia water

 Bluing reagents change the reddish-purple hematoxylin to a blue or purple-blue color. It is
a pH-dependent reaction and occurs in an alkaline solution.

E. Passing in changes of 95% alcohol and then changes of xylene to complete the staining process
 After thorough de-waxing, the slide is passed through several changes of alcohol to
remove the xylene, then thoroughly rinsed in water. The section is now hydrated so that
aqueous reagents will readily penetrate the cells and tissue elements.

2. What is blueing? “Lake” in staining?

 “Blueing” is used to describe the procedure wherein the alum hematoxylin stained sections are
passed on to an alkaline solution which neutralizes the acid and free OH group that results in an
insoluble blue aluminum hematin-tissue-lake.