BLOCK PREPARATION.pptx

Block Prepration

1. BLOCK PREPARATION CUTTING AND STAINING OF SLIDES PRESENTED BY DR. POONAM

2. STEPS Embedding Trimming Cutting Staining Mounting

3. Embedding 1. Place tissue cassette in melted paraffin (65 degree) 2. Fill mold with paraffin 3. Place tissue in mold 4. Allow to cool (30 mint)

4. MEDIAS FOR EMBEDDING Paraffin wax – Routine in use Resins – electron microscopy Agar – for several friable tissue , Gelatin –lower melting point , frozen sectioning Celloidin – CNS tissue

5. EMBEDDING STATION

6. EMBEDDING STATION

7. Sectioning A . Rotary microtome 1. 5-10 mm 2. resolution vs. staining B. Cryostat C. Freezing microtome D. Vibratome 1-1

8. Leica RM2125RT

9. REQUIREMENTS Water bath 56-58 degree – DW, high concentration of alcohol, trace of detergent can be added to water beneficial in flattening sections. Hot plate Pointed forceps Brush Scalpel Slide rack

10. Continued Sections adhesives - Albumin , gelatin, starch, cellulose, poly-L- lysin Routinely used in brain, spinal cord, blood clot , decalcified tissue .

11. Sectioning – Trimming the Blocks Old knife or blade to be used Setting the thickness to 15 µm

12. Cutting sections After trimming suitable areas of tissue is exposed for cutting Placed on melting ice for some minutes It gives similar consistency Small amount of water absorbed , swelling it and making sectioning easier Cut at 3-4µm Strokes to be regulated Ribbons of sections are convenient. Sections put in oven 50-60 degree for drying

13. Sectioning 1 . Place tissue block in microtome with wide edge of trapezoid lowest, and parallel to knife 2. Advance blade toward block 3. Begin sectioning

14. Mounting sections A . 40 o C water bath 1. Flattens paraffin section 2. Permits mounting on slide B. Gelatin & albumin C. Glass slides D. Oven / air dry

15. Staining A. Basic dye: hematoxylin 1. basophilic structures: DNA, RNA 2. differentiation: sodium bicarbonate B. Acid dye: eosin 1. acidophilic ( eosinophilic ) structures a. mitochondria, collagen C. Water soluble dyes (paraffin sections) D. Clearing agent (remove paraffin) E. Rehydrate F. Stain (trial & error timing)

16. Routine H& E staining Hematoxylin is extracted from heart wood Oxidation product hematin , natural dye is responsible for color property Prepared by natural or chemical oxidation Types – Harris hematoxylin (mercuric oxide) Mayer’s hematoxylin (sodium iodate )

17. Harris hematoxylin Hematoxylin crystals: 5gm Alcohol, 100% :50.0 ml Ammonium or potash alum : 100gm Distilled water : 1000.0 ml Mercuric oxide : 2.5 gm Glacial acetic acid : 40 ml

18. METHOD Stained dissolved in alcohol Add alum already dissolved in warm DW Mixture is brought to boil Slowly add mercuric oxide On cooling add acetic acid It gives selective and precise staining of nuclei

19. Staining Procedure De wax section in xylene , hydrate through graded alcohol to water. Rinse in running water Stain in hematoxylin for 4 to 5 minutes. Rinse in running water for 2 to 3 minutes Differentiate in 1% acid for 4 to 7 dips. Rinse in running water for 5 to 6 minutes. Ammonia 1 % 5 to 7 dips. Rinse in running water for 5 to 6 minutes. Stain in eosin 1% for 5 to 6 minutes. Rinse in running water for 1 to 2 minutes. Dehydrate In 3 changes of alcohol : 2 mins each. Dehydrate In 3 changes of xylene : 2 mins each. Mount with DPX.

20. Result Nuclei: blue-black Cytoplasm: Varying shades of pink Muscle Fibres : Deep pinky red RBCs: Orange/ red Fibrin: Deep pink

21. Automated Staining 1. Slide rack 2. Solutions a. rehydration b. stain c. dehydration

22. Coverslipping A. Coverslip & mounting medium (not miscible with water) B. Dehydrate C. Clearing agent D. Permount