Histopathology

Basic procedure of histopathology in lab

1. Histology: Lab Manual Dr. M andeep Kaur PGGCG, Sector 42, Chandigarh [email protected]

2. Requirements Chemicals Ethanol Formalin Benzene Xylene Paraffin wax DPX H & E stain HCl & NH 3 Glassware/instruments Coplin Jars Glass slides Beakers Wax dispenser Hot plate Water bath Rotary microtome L-shaped moulds Oven Microscope

3. Histology procedure Fixation Dehydration Clearing Infilteration Embedding Section cutting Staining Mounting

4. Histology Procedure Fixation

5. Tissue Fixation Tissues are fixed: To preserve cells and tissues constituents To prevent their degradation Bouin’s fixative/formalin for 24 hrs (with fixative’s volume 20 times greater)

6. Histology Procedure Fixation Tissue Processing Dehydration Clearing Infilteration

7. Dehydration 30% 50% 70% 70% 90% Ab Alcohal 3 min 3 min 3 min overnight 1 hr in each grade Remove fixative and water from the tissues and replacing with dehydrating fluid.

8. Clearing Replacement with benzene The tissues were shifted to varying concentrations of benzene and alcohol in order to replace alcohol with benzene: Absolute alcohol: Benzene:: 1:1 for 1 hr Absolute alcohol: Benzene:: 1:3 for 1 hr Pure benzene for 1 hr Replacement of dehydrating agent with a fluid that is totally miscible with both the dehydrating fluid and the embedding media.

9. Benzene: wax: 1:1 for 30 min Benzene: wax: 1:3 for 30 min Immersed in molten paraffin wax & two changes are given in 1 hr interval Replacement with wax Benzene from tissues was then replaced by wax as follows: Infilteration Replacement of clearing agent with embedding media.

10. Histology Procedure Fixation Tissue Processing Dehydration Clearing Infilteration Tissue embedding

11. Embeddin g Suitable sized tissue was embedded in molten wax in L- shaped moulds and rectangular wax blocks are prepared. Tissues are orientated in desired direction. provide sufficient external support during sectioning

12. Histology Procedure Fixation Tissue Processing Dehydration Impregnation Clearing Tissue embedding sectioning

13. Sectioning 5 µ thin sections are obtained using rotary microtome. Stretching Sections are fixed on glass slides with a very thin coat of albumin and stretched on hot plate using water. Dewaxing The sections on slides were dewaxed in xylene and passed through descending series of alcohol and finally in water (2-3 min in each). 30% 50% 70% 90% Water

14. Histology Procedure Fixation Tissue Processing Dehydration Impregnation Clearing Tissue embedding sectioning staining

15. Staining Sections are stained with H aematoxylin for 15 min. Differentiated in 1% HCl and NH 3 solution for 2 min. washed with water. Sections were dehydrated in ascending series of alcohol reaching upto 90% alcohol (2-3 min in each grade). Sections were stained with Eosin for one min and differentiated in 90% alcohol and dehydrated in absolute alcohol (2-3 min).

16. Histology Procedure Fixation Tissue Processing Dehydration Impregnation Clearing Tissue embedding sectioning staining Mounting Viewing

17. Sections were passed through xylene for clearing wax and mounted in DPX

18. Histopathological studies of kidney Fig: Photomicrographs (H & E) of Kidney of Ctenopharyngodon idellus Abbreviations: DCT-Distal convulated tubule, PCT- Proximal convulated tubule, BC-Bowman’s capsule, CD-Collecting duct

19. Thank you