PREPARED BY: DEBBRA MARCEL, VRI, 2011
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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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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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