Histopathology Embedding to Staining the Slides
- 1. LECTURE NO 3 Histopathology Part 2
- 2. Tissue Processing ManualTissue Processing Automatic Tissue Processing
- 7. Tissue Embedding Embedding is the process in which the tissues or the specimens are enclosed in a mass of the embedding medium using a mould. • Leuckhart's L pieces • Compound embedding steel Mould TwoTypes of Moulds
- 8. Compound embedding metal Mould
- 9. Compound embedding steel Moulds
- 10. L pieces Moulds
- 11. Tissue Blocks
- 12. Paraffin section cutting • Equipment required • 1. Microtome • 2.Water bath preferably thermostatically controlled • 3. Fine pointed forceps • 4. Small hair brush • 5. Clean glass slides • 6. Section adhesive
- 13. Sectioning Wax is removed from the surface of the block to expose the tissue. Blocks are chilled on a refrigerated plate or ice tray for 10 minutes before sectioning. A microtome is used to slice extremely thin tissue sections off the block in the form of a ribbon. The microtome can be pre-set to cut at different thicknesses, but most tissues are cut at around 5 µm.
- 14. Microtomes are mechanical devices for cutting uniform sections of tissue of appropriate thickness.
- 15. Ribbon Sections
- 16. The section thickness The appropriate level for routine purposes to 4-6 microns.
- 17. Water Bath set at 450C Once cut, the tissue ribbons are carefully transferred to a warm water bath set at 45ºC
- 18. Section adhesives • An adhesive is a substance which can be smeared on to the slides so that the sections stick well to the slides. • Types of adhesive • Albumin • Gelatin • Starch • Adhesive are either added to water bath or smeared thinly on slide.
- 19. Lifting and Drying of section The floating sections were stretched in the water bath and lifted carefully on clean glass slides which smeared with a thin film of egg albumin.The slides containing tissue section place in incubator for dying at 37ºC for 2 hours.
- 20. Hematoxylin and Eosin Hematoxylin reacts like a basic dye with a purplish blue colour. It stains acidic, or basophilic, structure including the cell nucleus (which contains DNA and nucleoprotein) and organelles that contain RNA such as ribosomes and the rough endoplasmic reticulum. Eosin is an acidic dye that is typically reddish or pink. It stains basic, or acidophilic, structures which includes the cytoplasm, cell walls, and extracellular fibers
- 21. Staining Procedure Dewax / Deparaffinization the paraffin sections in xylene 2 × 5 min each Rehydrate through descending concentrations of ethyl alcohol such as 3 minutes in 100,3 minutes in 90%,3 minutes in 70% ethyl alcohol respectively
- 22. Staining Procedure Wash in running tap water for 3 min Stain for 8-10 min in Hematoxylin Wash in running tap water for 3 min Decolorize briefly in 1% acid alcohol for 2 s ?
- 23. Acid Alc0hol Acid alcohol is a differentiation reagent. It is used in hematoxylin eosin (HE) staining and provides excellent differentiation between nuclear and non-nuclear structures. Differentiation rinses dyes from cytoplasm while the nucleus remains stained.That occurs because the nuclear dye bonds stronger to the nucleus than to the cytoplasm. 1ml Conc HCL and 99ml 70% ethanol
- 24. Staining Procedure Wash in running tap water for 1 min Then 2 dips were given in Ammonia water in ordered to restore blue color of nuclei means, bluing the hematoxylin stain Wash in running tap water Stain for 2–5 min in eosin (counter stain) ?
- 25. The Bluing Step One of the steps in the H&E procedure is bluing. This step converts the initial soluble red color of the hematoxylin within the nucleus to an insoluble blue color Ammonia water Dilute lithium carbonate
- 26. Staining Procedure Dehydrate it through ascending order of ethyl alcohol concentrations as in 70%, 90% and in 100% for 1 minute each The sections on slides were immersed in Xylene (Clearing) by giving two dips in pure Xylene solution.
- 27. Mounting of Slide By DPX Histological sections, which need to be examined for any length of time or to be stored must be mounted under a cover-slip.
- 28. Ready to Observe Under the Microscope The stained slides are then examined under 10X and 40X magnifications for observing prominent lesions.
- 29. Slide Preparation • https://www.youtube.com/watch?v=nUjK4n3_1C8 • https://www.youtube.com/watch?v=7-LIbAWPc-g