Histopathological Approach to Rat Liver Tissue
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Procedure
- After deep ether anesthesia, dissect the rat’s liver (Wistar albino rats, 200 � 250 g) by cutting on the ventral side.
- Fix 2 � 3 mm. of the liver tissue in neutral buffered formalin (10% formaldehyde in Phosphate buffered saline) over night.
- 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.
- Then dip the tissues in acetone for a period of 1 � 2 h with periodical shaking.
- After removing the acetone, add xylene to check for the appearance of milkyness. If milkyness appears then repeat the dehydration procedure.
- Impregnate the dehydrated tissue in paraffin wax (m.p. = 56° C) for a period of 1h at 58 � 60 ° C.
- Pour the molten paraffin into L-block along with the tissues and allow it to become hard.
- Section the tissue into very thin (2�8 or 5 � 10 micrometer) sections using a microtome.
- 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.
- Place slides containing paraffin sections on a slide holder.
- Deparaffinize with Xylene for 20 � 30 minutes and blot the excess xylene.
- Rehydrate the tissue successively with 100%, 90%, 80% isopropyl alcohol for 2 � 3 min. each and put it into water for 3 min.
- Blot the excess water; put the tissue into Hematoxylin stain for 1 � 2 min.
- Remove it from Hematoxylin stain and then again put it into tap water for 1 � 2 min.
- 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.
- Immerse the tissue in Eosin stain for 30 secs.
- Dehydrate the tissue successively with 80%, 90%, 100% isopropyl alcohol and finally with Xylene for 20 � 30 min.
- Place coverslip on the slides using one drop of DPX, taking care to leave no bubbles and dry overnight to make the permanent slide.