Molecular Pathology Core Laboratory

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Molecular Pathology Core Laboratory

• PI ???/???
• Chin-Wen Chi, Ph.D., Anna F-Y. Li, M.D.
• Department of Medical Research Education
• Department of Pathology
• Taipei Veterans General Hospital
• National Yang Ming University

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Specific Aims

• 1.To establish a core facility for the
  preparation and use of pathological samples.
• 2.To provide pathological services for basic
  researchers.

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Service provided

• (1)Dr. ??? preparation of wax array blocks and 8
  sections per block (8 samples), preparation of
  array block (one block with 24 samples)
• (2)Dr. ??? immunostaining of 8 human hepatoma
  tissue array with 3 monoclonal antibodies.
• (3)Dr. ??? provided both normal ( 2 slides) and
  tumor (10 slides) tissue array slides (33-38
  tissues). provided both normal ( 10 slides) and
  tumor (10 slides) tissue array slides (9
  tissues).
• (4)Dr. ??? Wax block preparation (38 blocks),
  section and HE stain (2 blocks), with image
  files.
• (5)Dr. ??? Immunostaining of human hepatoma
  array slides (10 slides), with two new antibodies
  and their peptide blocker.

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      The PixCell II performs
Laser Capture Microdissection (LCM) from
heterogeneous tissue samples simply, quickly and
precisely. In minutes, you can locate a single
cell or large groups of cells and, using a simple
aim-and-shoot method, extract them for subsequent
molecular analysis. LCM preserves the exact
morphologies of both the captured cells as well
as the surrounding tissue. The PixCell II
transfers cells from paraffin-embedded and frozen
tissue samples, stained and immunolabelled
slides. Monitor and document the entire process,
and store images in the archiving workstation.
Microdissect fluorescently-stained cells with the
optional fluorescence package.    
 
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Applications utilizing LCM Research
Applications      Genomics           Differentia
l Gene Profiling           Loss of
Heterozygosity           Microsatellite
Instability           Gene Quantification
     Proteomics           Two-Dimensional
Protein Gels           Western Blotting
          Immuno-quantification of Proteins
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Molecular Pathology WorkshopPreparation and
Staining of Tissue

• ???/???
• Department of Medical Research Education
• Department of Pathology
• Taipei Veterans General Hospital
• National Yang Ming University

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?????????????

• Materials used in your project
• Cells.
• Tissues
• Target of your study
• DNA, RNA, protein
• .

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91.01.02 DOH
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?????????????

• Materials used in your project
• Non-human Cells or tissues rats, mice, rabbits,
  dogs, pigs, etc.
• Please get approval from your Institutes
  ?????????
• (???40248?? July 13, 2001)
• .

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????-FIXATION (1)

• What will happen to a tissue after removal from
  living condition?
• Bacteria growth
• Autolysis
• How to maintain cell or tissues structure, solid
  and liquid phases.
• .

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FIXATION (Time and Temperature)

• Tissue should be fixed ASAP after removal. If
  fixation can not be done right away, tissue
  should be kept in refrigerator until the fixative
  can be applied.
• RNAlater do not put into refrigerator, it works
  at room temperature..
• .

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Fixing solutions

• 1. Fast penetration
• 2. Coagulate cell contents into insoluble
  substances
• 3. Protect tissue from shrinkage and distortion
  during dehydration, embedding and sectioning.
• 4. Allow cellular targets for further assay such
  as staining or in situ hybridization.
• .

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Fixing solutions

• 1. Acetic acid
• 2. Acetone
• 3. Ethanol.
• 4. Formaldehyde
• 5. Osmium tetroxide
• 6. Picric acid.
• .

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FIXATIONspecial consideration

• Lipiodol uptake in hepatoma
• Aqueous or alcoholic fixative?
• In situ hybridization
• Formalin, paraformaldehyde, acetone.
• In situ perfusion fixation.
• Time of fixation, size of tissue.
• .

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?????? (1)

• ???? ????? ,?????????2 cm x 2 cm x 0.5
  cm??????(??????),??????15-20????4
  Paraformaldehyde??? , ????? 12-24??????10
  Formalin????.
• Cells, Soft vs. hard tissues.

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?????? (2)

• ???? , ?? , ?? ????5? , ????????????? ,
  ????????????????? , ??????8????????? .
  ????????????????
• (?????????? SHANDON ?? Hypercenter XP)
• 1. 70 alcohol 4 hrs
• 2. 80 alcohol 1 hr
• 3. 95 alcohol 1 hr
• 4. 95 alcohol 1 hr
• 5. 100 alcohol 1 hr
• 6. 100 alcohol 1 hr
• 7. 100 alcohol 1 hr
• 8. xylene substitute 1 hr
• 9. xylene substitute 1 hr
• 10. paraffin 1 hr
• 11. paraffin 2hr
• ???? ????8??????? , ?????????????????
  ,??????????????. ????5 mm ??H.E. stain??,
  ????????????

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• 1. 70 alcohol 1 hrs
• 2. 80 alcohol 1 hr
• 3. 95 alcohol 1 hr
• 4. 95 alcohol 1 hr
• 5. 100 alcohol 1 hr
• 6. 100 alcohol 1 hr
• 7. 100 alcohol 1 hr
• 8. xylene substitute 1 hr
• 9. xylene substitute 1 hr
• 10. paraffin 1.5 hr
• 11. paraffin 1.5 hr
• ???? ????8??????? , ?????????????????
  ,??????????????. ????5 mm ??H.E. stain??,
  ????????????

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?????? (3)

• ???? ????8??????? , ?????????????????
  ,??????????????. ????5 mm ??Hematoxylin and eosin
  stain??

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Tissue Array Paraffin Block
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O.C.T.??( TISSUE-TEK MILES Inc. 4583)

• ?x?x? 0.7 x 0.7 x 0.7 (cm3)???.
  ??????DMEM??????(GIBCO/BRL),??????,????????N?T,???
  ?????????????. O.C.T.???liquid nitrogen
  frozen??????-80????

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Sectioning
Cut a thin slice of tissue (5-8 um) 1. Frozen
section 2. Wax embedded tissue Slides coated
slides(Muto glass silane coated
slides) non-coated slides (LCM) Superfrost
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Sectioning
Sections of paraformaldehyde fixed, OCT-embedded
tissue are sectioned at 7 to 10 mm and can be
stored desiccated at -70C until needed.
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Cell plating on slides
To sterilize glass coverslips, dip in ethanol and
flame. One can use 22x22x1 mm3 coverslips and
put them in 6-well plates. Seed 100,000 cells
per well overnight and fix the next day.
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Cell plating on slides
Remove the media and rinse once with PBS. Remove
the PBS and immediately add -20C methanol. (Do
not allow the cells to dry.) Put the plate in a
-20C freezer for 5 min. Remove the methanol and
add PHEM buffer (25 mM HEPES, 10 mM EGTA, 60 mM
PIPES, 2 mM MgCl2, pH 6.9) Fixed cells are kept
at 4C in PHEM prior to immunostaining.
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Sectioning Handling RNA
Collection of Tissue for RNA Analysis
Ribonucleases (RNases) are very stable and
active enzymes that degrade RNA. Minute amounts
are sufficient to destroy RNA. It is important
to use RNase free conditions during all apsects
of tissue collection. Always wear latex or vinyl
gloves while handling reagents and RNA samples to
prevent RNase contamination from the surface of
the skin or from dusty laboratory equipment.
Change gloves frequently and keep tubes closed.
The use of sterile, disposable polypropylene
tubes is recommended.
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SectioningHandling RNA
Necrospy Instruments Metal instruments
(scissors, pick-ups, etc) can be autoclaved prior
to use or washed with DEPC water and baked at
240C for 4 or more hours.Autoclave only is not
enough. Glassware can be treated with 0.1 DEPC
(diethyl pyrocarbonate). Fill glassware with
DEPC, allow to stand overnight (12 h) at 37C,
and then autoclave or heat to 100C for 15 min to
remove residual DEPC. Alternatively, all plastic
and glass ware, instruments and work surfaces may
be treated with RNase Zapo (Ambion), a RNase
decontamination solution. Sample Collection of
Rodent or Non-human samples At necropsy,
tissues should be collected within 12-15 minutes
following death. No specific form of euthanasia
is required. If the tissue collection time is
much greater than 15 minutes, RNases will have
been activated and it is highly unlikely that
good quality RNA will be obtained for further
analysis.
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• 1. xylene substitute 10 min
• 2. xylene substitute 10 min
• 3. 100 alcohol 1 min
• 4. 100 alcohol 1 min
• 5. 95 alcohol 1 min
• 6. 80 alcohol 1 min
• 7. 70 alcohol 1 min
• 8. Running water 3 min
• 9. Harries Hematoxylin 15 min
• 10. Running water 2 min
• 11. paraffin 1.5 hr
• ???? ????8??????? , ?????????????????
  ,??????????????. ????5 mm ??H.E. stain??,
  ????????????

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• 11. 0.5 acid alcohol 3 sec
• 12. Running water 1 min
• 13. Ammonia water 30 sec
• 14. Running water 20 min
• 15. 0.5 alcoholic eosin 2-5 min
• 16. 95 alcohol 10 sec
• 17. 95 alcohol 10 sec
• 18. 100 alcohol 30 sec
• 19. 100 alcohol 1 min
• 20. Xylene substitute 5 min
• 21. Seal the slides with sealer
• 11. paraffin 1.5 hr
• ???? ????8??????? , ?????????????????
  ,??????????????. ????5 mm ??H.E. stain??,
  ????????????

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Mounting media

• Organic mountant Rapid mounting media for
  microscopy (Merck), or Permount
• Aqueous mountant Kaisers glycerol gelatin
  (Merck). (Intracellular components such as lipid
  will be dissolved by organic mountant)
• Watch out for air bubbles.

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Useful website and book

• http//dir.niehs.nih.gov/dirlep/
• http//dir.niehs.nih.gov/dirlep/immuno.html
• P53, COX-2, Insulin, PCNA, BrDU, ER, GST-pi,
  ..etc (images and protocols)
• Laser capture microdissection protocols.
• Animal Tissue Techniques, GL Humason
• W. H. Freeman and Company. (????)

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Reagents

• Paraformaldehyde Nacalai
• Hematoxylin, 1 Eosin Y solution MUTO Pure
  Chemicals Co.
• Xylene substitute Ultra Clear from J. T. Baker.
• Kaisers glycerol gelatin, Rapid Mounting media
  for microscopy from Merck.

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Acknowledgement

• 1. National Yang Ming University
• Genome Center.
• 2.Departments of Surgery(General Surgery,
  Experimental Surgery), Pathology, and Research
  Education, Taipei Veterans General Hospital.
• 3. National Health Research Institute