This document outlines the basic steps of tissue processing for microscopic examination: embedding, sectioning, staining, and mounting. Tissue is embedded in paraffin wax and allowed to cool, then sectioned on a microtome. Sections are mounted on slides and stained using hematoxylin and eosin (H&E). Hematoxylin stains cell nuclei blue while eosin stains cytoplasm and extracellular components pink. After staining, sections are dehydrated and coverslipped using a mounting medium. The overall process prepares tissue for microscopic analysis and diagnosis.

1. 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. 40o 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