PREPARED BY: DEBBRA MARCEL, VRI, 2011

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1. Post Mortem 2. Fixation 3. Tissue Trimming 4. Tissue Processing 5. Embedding 6. Sectioning 7. Slide preparation 8. Staining 9. Mounting 10.Observing

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A) Cut carcass to get the necessary organ according to the case/ intended test (to be done ASAP) to minimize post-mortem changes

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Organ fixed into 10% formalin solution at least 24hr before proceed to the next step -To maintain the morphology/structure of tissues as per received. Other fixation agent = Zenker’s solution & Bouin’s solution

*CAUTION: CARCINOGENIC!!

Hypotonic=pH:6.8

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a) Before the tissue is trimmed, the fixed organ washed under running tap water to get rid of the formalin residue

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b) Trimming organs into tissues ( >3mm thickness) • While selecting tissue, a brief description of the nature of tissue & site of origin should be recorded appropriately • Choose the lesion & non-lesion part for easy observation

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a) The trimmed tissue inserted into the labeled tissue cassette (plastic embedding cassette) & washed once again before proceed to tissue processing

* The remaining organ/tissue restored into the jar containing 10% formalin solution where it was placed before.

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Histokinette (Automatic Tissue Processor)

Process (3 main process)

70% alkohol 1-2 jam Alkohol 1 1-2 jam Alkohol 2 1-2 jam Alkohol 3 1-2 jam Alkohol 4 1-2 jam Alkohol 5 1-2 jam Alkohol 6 1-2 jam Alkohol 7 1-2 jam Chloroform 1 1-2 jam Chloroform 2 1-2 jam Wax 1 1-2 jam Wax 2 1-2 jam (vacuum bath) Next, tissue is ready to the next step… “embedding”

Dehydration

Clearing

Wax Infiltration

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a.k.a “The tissue waxing process”

Tissue cassettes then placed inside aluminum metal tray on hotplate containing melted crystal wax *crystal wax melting point=54-56°c

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Then tissues is taken out from cassettes & transferred into a stainless steel base mould

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From hotplate, the mould is then meticulously transferred onto the coldplate , topped with crystal wax until almost full & covered with cassette

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After several minutes, the frozen waxed tissues is then taken out from the mould

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TARAA…

The tissue blocks is done!

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The tissue block is soaked in an iced cold distilled water to densify the block

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The frozen tissue block is then placed onto the rotary microtome specimen clamp necessarily

**extra careful should be given while handling the disposable microtome blade (extremely sharp)

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Adjusting hand-wheel & cutting thickness (in this case, we had set to 5µm cutting )

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Frozen waxed tissue meticulously cut using rotary microtome by making 2 or 3 copies per block (so that when one of the tissues accidently ruined, we’ll have some spares for it!)

Block should be trimmed first before cutting section

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The fine layer of waxed tissue is then laid onto a warm waterbath (40°c-42°c) to remove wrinkles

FLOATING THE FROZEN SECTION

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Adhesived slides – glass slide should be coated with albumin solution before placing the tissue section onto the slide

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Placing the tissue section onto the slide carefully • Submerge the glass slide 90° then emerge the slide from the tissue section underneath • Make sure the section is not crumple

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Draining slides for a few minutes then place into the incubator at 37°c at least for ½ hour

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6 5 4 3 2

9 8 7 10 11 12

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H&E STAINING (ROUTINE STAINING)

1. Xylene 1 2. Xylene 2 3. Alcohol (70%) 4. Alcohol (90% ) 5. Alcohol (100%)

6. Hematoxylin

7. Eosin

8. Alcohol (100%) 9. Alcohol (90%) 10. Alcohol (70%) 11. Xylene 3 12. Xylene 4

to de-wax tissue (1-2 mins.)

to get rid of xylene oily properties (1-2 mins.)

to colourize (blue=base)the nucleus (15 mins.)

to colourize (red=acid)the cytoplasm (5 mins.)

to dry out the wet tissue (1-2 mins.)

to clearing the tissue (1-2 mins.)

WASH IN CONTAINER (UNDER RUNNING TAP WATER)

WASH IN CONTAINER (UNDER RUNNING TAP WATER)

WASH IN CONTAINER (UNDER RUNNING TAP WATER)

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1 2

Mount the slide with mounting glue (e.g synthetc resin medium) & cover with coverslip

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The slide is now ready to be observed under microscope ? X objective

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Light microscopes

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