Immunohistochemistry

2. Xylene (Gadot, Haifa, Israel; cat. no. 1330-20-71).

3. Absolute ethanol (see Subheading 2.3., item 6).

4. Microwave (Electrolux supplemented with probe).

5. 3% citrate buffer (pH 6.1). Prepare 0.1 M citrate acid solution: 1.921 g citric acid (FRUTAROM; cat. no. 55110) in 100 mL double-distilled H2O. Mix 9.1 mL with 40.9 mL 0.2 M Na2HPO4 (Merck; cat. no. 10686), add double-distilled H2O to 100 mL.

6. 3% hydrogen peroxide (H2O2) (Merck, cat. no. 107209): dilute 3 mL in 97 mL methanol (FRUTAROM; cat. no. 55562380).

7. PBS Dulbecco's solution (Invitrogen Corporation; cat. no. 14190-094).

8. Non-immune goat serum blocking solution (Histostain-SP kit; Zymed Lab Inc., San Francisco, CA; cat. no. 95-6543B).

9. Antibodies: rabbit polyclonal anti-GFP (1:2000) (Molecular Probes, Eugene, OR; cat. no. A-6455), mouse monoclonal anti-human CD34 (1:50) (DakoCytomation, Glostrup, Denmark; M-7165), rabbit polyclonal anti-mouse CD31 (1:2000) (kindly provided by J. Mardi, Yale University, New Haven, CT).

10. Secondary antibodies: goat anti-rabbit (Zymed Lab Inc.; cat. no. 50-235), anti-mouse biotinylated secondary antibody (Histostain-SP kit; Zymed Lab Inc.; cat. no. 95-6543B).

11. Pre-immune rabbit (Zymed, Lab Inc.; cat. no. 50-061) or mouse (Zymed, Lab Inc.; cat. no. 50-235) sera (for negative controls).

12. AEC-substrate chromogen kit (Zymed, Lab Inc.; cat. no. 00-2007).

13. Hematoxylin solution (Merck; cat. no. 4305).

2.7. Extraction of RNA From Teratomas, Teratomas Bearing Tumors, and From Tumors

1. Dissecting tools (see Subheading 2.5.1., item 1).

2. Scalpel (size 10; Albion Surgical Ltd., Sheffield, England; cat. no. 1531746).

3. 50-mL sterile tubes (Greiner, Bio-One; cat. no. 227270).

4. Sterile pipets, individually wrapped (Greiner, Bio-One; cat. no. 606180).

5. Tri-reagent (Molecular Research Center Inc., Cincinnati, OH; cat. no. TR-118).

6. Homogenizer POLY TRON 2100 (Kinematica, Lucerne, Switzerland).

8. DEPC-H2O (Biological Industries; cat. no. 01-852-1A).

9. 1-bromo-3-chloro-propane (Sigma-Aldrich; cat. no. B-9673).

10. Centrifuge (Sorval, Asheville, NC; model no. RC5C).

11. Isopropanol (FRUTAROM; cat. no. 5553120).

13. RNAsin (Promega, Madison, WI; cat. no. N-211A).

2.8. Removal of Genomic DNA From RNA Samples

2. H2O saturated phenol solution (Biological Industries; cat. no. 01-852-1A).

3. Chloroform: isoamyl alcohol (24:1) (Bio-Lab; cat. no. 03072301).

5. Micro-Centrifuge 5417R (Eppendorf, Hamburg, Germany).

7. DEPC-H2O (Biological Industries; cat. no. 01-852-1A).

9. Spectrophotometer UV/Visible Ultraspec 200 (Pharmacia Biotech, Piscataway, NJ).

2.9. Molecular Analysis of Gene Expression Pattern in Teratomas Bearing Tumors

1. QuantiTect SYBR Green Reverse Transcription Polymerase Chain Reaction (RT-PCR) kit (QIAGEN Inc.; cat. no. 204243).

2. Rotor-gene 2000 (Corbett Research, Sidney, Australia).

3. Methods

3.1. Establishing Cancer Cells Stably Expressing EGFP

1. Seed 104-5 x 105 cells per 10-cm tissue culture dish (depending on the size and the proliferation rate) in the appropriate culture media.

3. Remove culture media and add fresh media.

4. Mix 95 ||L media with 5 ||L FuGENE6 transfection reagent.

5. Incubate for 5 min at room temperature.

6. Add 1 | g pEGFP-N1 vector DNA (transfection grade) dissolved in H2O, mix and incubate for an additional 15 min at room temperature.

7. Add mixture to the media in the culture dish and mix well.

8. Incubate for 48 h in the appropriate cell growing conditions.

9. Examine the transfection efficiency in the culture dish using an inverse fluorescence microscope.

10. Remove the culture media, wash cells once with PBS, add trypsin, incubate at 37°C for 5 min, and collect the cells.

11. Split the cells into 10-15 x 10-cm dishes (depend on the transfection efficiency).

12. Add 300 |g/mL G418 into the growing media for selection of stably Neoresistance gene expressing cells (that also express the EGFP).

13. Transfer single resistant colonies that express EGFP into a 24-well plate and continue propagating the cells.

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