Histopathological Approach to Rat Liver Tissue

Procedure

1. After deep ether anesthesia, dissect the rat’s liver (Wistar albino rats, 200 – 250 g) by cutting on the ventral side.

2. Fix 2 – 3 mm. of the liver tissue in neutral buffered formalin (10% formaldehyde in Phosphate buffered saline) over night.

3. After fixation, place the tissues in 70% isopropyl alcohol for 3 hours and then in each ascending strength (80%, 90%, 100% isopropyl alcohol) for 2 hours each. The amount of alcohol used should be 15 times of the size of the tissue.

4. Then dip the tissues in acetone for a period of 1 – 2 h with periodical shaking.

5. After removing the acetone, add xylene to check for the appearance of milkyness. If milkyness appears then repeat the dehydration procedure.

6. Impregnate the dehydrated tissue in paraffin wax (m.p. = 56°C) for a period of 1h at 58 – 60 °C.

7. Pour the molten paraffin into L-block along with the tissues and allow it to become hard.

8. Section the tissue into very thin (2–8 or 5 – 10 micrometer) sections using a microtome.

9. Mount the tissue on the slides with Mayer’s albumin solution (a mixture of equal parts of egg white and glycerin, beaten and filtered with the addition of 1% sodium salicylate) and keep in warm oven for 2 h at 60°C.

10. Place slides containing paraffin sections on a slide holder.

11. Deparaffinize with Xylene for 20 – 30 minutes and blot the excess xylene.

12. Rehydrate the tissue successively with 100%, 90%, 80% isopropyl alcohol for 2 – 3 min. each and put it into water for 3 min.

13. Blot the excess water; put the tissue into Hematoxylin stain for 1 – 2 min.

14. Remove it from Hematoxylin stain and then again put it into tap water for 1 – 2 min.

15. Dip the slides containing tissue sections into 1N HCl followed by Scott’s water ( Sodium Bicarbonate 3.5 g, Magnesium sulphate 20 g, distilled water 1 litre ) for 1 min each.

16. Immerse the tissue in Eosin stain for 30 secs.

17. Dehydrate the tissue successively with 80%, 90%, 100% isopropyl alcohol and finally with Xylene for 20 – 30 min.

18. Place coverslip on the slides using one drop of DPX, taking care to leave no bubbles and dry overnight to make the permanent slide.