Abstract:
Staining is an integral part of modern histopathology and without proper staining
histology would be a mere concept. Haemotoxylin and Eosin (H&E) stain have been
evolved during last several decades and considered as the golden standard for histological
examinations for paraffin or resin embedded animal tissues which have been fixed,
processed, embedded and sectioned. It has been widely used and has recognized as the
best method to highlight the fine structure of cells without compromising the morphology
of the cell. Among many synthetic aniline dyes Haemotoxylin is the only natural dye which
is an extract of the Logwood (Haematoxylum campechianum). After many attempts of
using dyes to stain animal tissues such as ovary from Sus scorfa domesticus which is
inevitably rich with large fat globules, it has been discovered that traditional methods
were not very effective. The objective of current study was to optimize existing H&E
staining to achieve better results in high fat cultured tissues than low fat fresh tissues.
Briefly, following procedures were adapted with strict time control methods.
Deparaffinization of the sections on glass slides was performed. Two dips in xylene for 4
min each and followed by rehydration of the sections with a series of submerge in Ethanol
(ETOH). Starting from 100% ETOH two dips followed by 90%, 70%, 50% alcohol and
rehydration was completed with a final two dip of distilled water. All these steps were
done for 2 min each. Subsequently sections were submerged in Haemotoxylin for 15 min
followed by gentle washes with running tap water for another 10 min. Then all slides
were subjected to another dip in Eosin for 30 sec. Slides were washed gently again for
further 2 min with running tap water. Finally all the slides were dipped at 1-2 sec in 70%,
90%, 100% alcohol serial and xylene respectively. As soon as completion of the last xylene
step, cover slips were placed on top of the permount mounting medium which are already
placed on the tissue section and allowed the slides to dried-up for 24-36hrs before
microscopic observation. In conclusion, it has been discovered double staining of high fat
cultured tissue with Haemotoxylin and Eosin with this modified technique has evident
best microscopic observations, provided example images showing results produced using
modified protocol, as well as commentary on the strengths and limitations of the
approach. This study was supported by Sabaragamuwa University Research Grant,
SUSL/RG/2015/06.
