Histopathology Lab - Staining

Nadiah Bte Sukma

0705365E


Hi......

very sorry for the late post..


STAINING

In histopathology lab, all slides have to be stained using Haematoxylin & Eosin staining. Special stains request will be made after pathologists have seen the H&E slides and decided that the case needs further investigation with different stains eg PAS, GMS, RETIC.

The slides that ready for staining have to be initially air dried by placing on the sides of the hot plate until the wax shows signs of melting. this step is important because placing them directly on the hot plate will create a pool of water and melted wax (caused by sudden change of temperature) on the slide and hence, tissues might float off and orientation of tissues would change.

The dried slides are then placed on the hot plate with sections facing up for about 3 mins. Alternatively, can place into the drying oven at 99C for about 10 mins. This is to ensure that the tissue sections are fixed onto the slides so that it would not float off during staining. The heated slides are then manually loaded onto the automated staining machine.

The principle of H&E is to demonstrate the tissue constituents in contrasting colours. Haematoxylin is oxidized to haematein in the presence of an oxidising agent. Haematein is taken up by nucleic acids present in the nucleus in the presence of a mordant. The mordant used is usually a metal that is able to chelate the haematein dye to the tissue component. Eosin stains the cytoplasm and connective tissue pink.



The staining progame is as follows:

1. Xylene (2 mins)
2. Xylene (2 mins)
3 Absolute alcohol (1 min)
4. 95 % alcohol (1 min)
5. 70 % alcohol (30 secs)
6. Running water (30 secs)
7. Haematoxylin (3 ½ mins)
8. Haematoxylin (3 ½ mins)
9. Running water (30 secs)
10. 0.5 % acid alcohol (2 dips)
11. Running water (30 secs)
12. Lithium carbonate (30 secs)
13. Running water (30 secs)
14. Blue in running water (3 mins)
15. Eosin (25 secs)
16. 70 % alcohol (1 min)
17. 95 % alcohol (1 min)
18. Absolute alcohol (1 min)
19. Absolute alcohol (1 min)
20. Absolute alcohol (1 min)
21. Absolute alcohol (1 min)
22. Xylene (1 min)
23. Xylene (1 min)
24. Xylene (1 min or until ready for cover slipping)
25. Depex (using automated machine or manually)


Results:

Nuclei: Blue
Cytoplasm: Pink



The picture above shows and example of H&E staining at 4X maginification. The pictures shows the presence of cracks and folds which may interrupt with the diagnosis. If there are too many faults present, the tissue have to be re-cut and produce another new slide to be stained.