Department of Pathology
Yamaguchi University Graduate School of Medicine
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protocols






PROTOCOL FOR MANUAL TISSUE MICRODISSECTION

This method is applicable to various kinds of cancer tissues to reduce stromal tissue components from cancer specimens. Investigators should also expect to invest time initially by practicing on 10 to 20 cases to begin to feel comfortable with the technique. As they become skilled in the microdissectiion technology, they obtain tissue fragments rich in cancer cells in a short time.

I. Preparation of frozen tissue sections
1.  Set the machine (cryo-stat) for -20Ž
(It takes at least a few hours to get the optimal temperature. Fortunately, the machine is usually maintained at -20C in this laboratory.)
 
2. Squeeze one drop of OCT compound (Tissue Tek) in bottom of mold, then put @@the tissue specimen in the center of it, and full the mold by OCT compound. @@@(avoid to trapping air bubbles)
*Embed fresh tissues carefully in OCT in plastic mold, taking care not to trap air bubbles surrounding the tissue. Freeze tissue by setting mold on top of liquid nitrogen until 70-80% of the block turns white and then put block on top of dry ice. The frozen blocks may be stored at minus 80‹C for long-term@storage.
3.  Put mold in machine for 20-30 minutes.
This process can be omitted, when the tissue specimens frozen by another method are used.
4.  Take out the cylinder from machine and placed one drop of compound
   on up of fit for mold fixation, Convert the mold and put in top of the 
   cylinder, leave it for 10 minutes in the machine.
 
5. Remove moldfs frame (plastic).
6. Adjust the cylinder for tissue sectioning.
7. Cut the tissue block with a razor at 5ƒΚm, and put on the slide nicety.
 (A tissue section is prepared for microscopic observation. When the density of cancer cells is very low, another tissue specimen should be used.)
8. Then, section the tissue block at 30ƒΚm.
9. Make totally 12-16 slides with repeat of procedures No. (7 to 8 ).
10. Leave slides in Ethanol 100% for 5minutes.
 
11. Stain tissue sections with HE.
The lower concentration of hematoxylin and eosin may improve macromolecule recovery.
 
12. Arrange slides on the map-wood, and put a cover glass only on 5ƒΚm- 
Sections.
We should evaluate the cancer cell content in the tissue section before beginning microdissection. 

II. Tissue microdissection - Picking up the cancer cells
We perform microdissection on a standard inverted microscope (stereomicroscope) using a 27 gauge needle on a syringe as the microdissecting tool.
Prepare in advance;        ‡@1.5ml-eppendorfe tube
                         @@@@ ‡A70%-ethanol
                  @@@@@@@@‡B1ml-syringe with 27G needle
1.  At first, spill a little of Ethanol on the slide, look for cancer cell
   nest, and take out that by needle of syringe, put it in the eppendorf
   tube (tube with 1/2 volume alcohol).
While viewing the tissue through the microscope, the cell population of interest should be gently scraped with the needle. (As another way, first stromal tissues are removed from cancer tissue specimens, and cancer cell populations are left on the slide.) The dissected cells will become detached from the slide and form small dark clumps of tissue that can be collected on the needle. Several small tissue fragments can be procured simultaneously. Collection of an initial fragment on the tip of the needle will assist in procuring subsequent tissue. The tip of the needle with the procured tissue fragments should be carefully placed into a small PCR tube containing ethanol. Gentle shaking of the tube will ensure the tissue detaches from the tip of the needle.
2.  Centrifuge the tube for 5minutes at 14,000rpm.
3. Remove the alcohol only, soak the tissue fragments in PBS, and centrifuge the @tube for 5 minutes at 14,000rpm.
 
4. Remove PBS.
 
5.  Go to the DNA extraction step (Dneasy tissue kit, QIAGEN, or SepaGene, @@@Sankojunyaku Co., Ltd.)
 
DNA EXTRACTION FROM MICRODISSECTED TISSUE SPECIMENS
DNA extraction is performed with a DNA extraction kit according to the manufacturerfs instructions. We use two kits (products of Sanko-junyaku and Quagen) for DNA extraction.


‚s‚n‚o



@@FISH on formalin-fixed paraffin-embedded tissue section

Day 1

1. 6% neutral-buffred formalin fixation

2. Paraffin-wax embedment

3. Prepare 4-6 ƒΚm tissue section slide

4. Xylene 10 min X3

5. Ethanol 10 min X2

6. Microwave-heating for 15 min (0.01M sodium citrate buffer, pH
@@6.0)¨H2O (approximate 2 min)¨PBS (approximate 2 min)

7. 0.05%-0.5% pepsin/0.1N HCl  at 37Ž for 12 min.

8. H2O (approximate 2 min) X2

9. 70% Ethanol (2 min)¨85% Ethanol (2 min)¨100% Ethanol (2 min)¨air dry

10. Preheating of section slide (place the slide on the lid of water
@@bath at 73Ž before preparation and denaturation of probe solution)

11. Prepare probe solution (for example: 7ƒΚl hybridization buffer + 1ƒΚl
@@probe + 2 ƒΚl DDW for CEP probe, Vysis)

12. Denature probe solution at 73Ž for 5 min then move to 45Ž

13. Denature the section in a denaturation solution at 73Ž for 5 min.

14. 70% Ethanol (2 min at -4 to -20Ž)¨85% Ethanol (2 min at -4 to
@@-20Ž)¨100% Ethanol (2 min at -4 to -20Ž)¨air dry

15. Hybridization at 37Ž¨ cover and seal the section with coverslip and
@@rubber cement

16. Mark on the section slide

17. Incubation at 37Ž for 3 days

Denaturation solution
  7ml 20X SSC
14ml H2O
49ml formamide
 

Day 4

1. Remove rubber cement from slide and immerse in 2X SSC/0.3% NP-40 at
@@room for 2-5 minutes.

2. 2X SSC/0.3% NP-40 at 73Ž for 2 minutes

3. 2X SSC/0.3% NP-40 at room for 1 minute

4. 70% Ethanol (1 min)¨85% Ethanol (1 min)¨100% Ethanol (1 min)¨air dry

5. Counterstain and apply coverslip¨observation under fluorescence
@@microscope.

2X SSC/0.3% NP-40
4ml 20X SSC
36ml H2O
120ƒΚl NP-40



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