158
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
Pretreatment of
slides
tissue sections and spotted cells is significantly facilitated by using poly-Llysine coated glass slides ( described in Subprotocol 7).
For variations of these protocols, that may be important for specific applications the reader is referred to the Iiterature (see for example example
Manuelidis 1985; Scherthau and Cremer 1993; Rawlins et al. 1991; Chumakov et al. 1992; Klinger et al. 1992; Stocket al. 1993).
Materials
Poly-L-Lysine stock solution: 0,1 mglml poly-L-lysine (Mol. Wt. 70.000150.000) dissolved in water. Keep aliquots frozen at -20°C.
Procedure
1. Immerse glass slide in stock solution of poly-L-lysine and incubate for 5
min. Take care that the solution does not dry!
2. Wash 5 times with distilled water.
3. Air dry and store at room temperature. The slides can be stored for several weeks.
Subprotocol 8
Preparation of Single Cell Suspensions from Solid Tumor Material
Materials
• 70% ethanol ( -20°C).
• Poly-L-lysine coated slides
• Phosphate-buffered saline (PBS)
• Digestion solution: Pepsin from porcine starnach mucosa (2500 - 3000
units/mg). Dissalve 50 - 400 Jlg pepsin/ml in 0.01 M HCl
• 1% paraformaldehyde, dissolved in 0.1 M phosphate buffer (pH 7.2-7.4)
containing 5 mM MgCh
• 70%, 90% and 100% ethanol
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
Pretreatment of
slides
tissue sections and spotted cells is significantly facilitated by using poly-Llysine coated glass slides ( described in Subprotocol 7).
For variations of these protocols, that may be important for specific applications the reader is referred to the Iiterature (see for example example
Manuelidis 1985; Scherthau and Cremer 1993; Rawlins et al. 1991; Chumakov et al. 1992; Klinger et al. 1992; Stocket al. 1993).
Materials
Poly-L-Lysine stock solution: 0,1 mglml poly-L-lysine (Mol. Wt. 70.000150.000) dissolved in water. Keep aliquots frozen at -20°C.
Procedure
1. Immerse glass slide in stock solution of poly-L-lysine and incubate for 5
min. Take care that the solution does not dry!
2. Wash 5 times with distilled water.
3. Air dry and store at room temperature. The slides can be stored for several weeks.
Subprotocol 8
Preparation of Single Cell Suspensions from Solid Tumor Material
Materials
• 70% ethanol ( -20°C).
• Poly-L-lysine coated slides
• Phosphate-buffered saline (PBS)
• Digestion solution: Pepsin from porcine starnach mucosa (2500 - 3000
units/mg). Dissalve 50 - 400 Jlg pepsin/ml in 0.01 M HCl
• 1% paraformaldehyde, dissolved in 0.1 M phosphate buffer (pH 7.2-7.4)
containing 5 mM MgCh
• 70%, 90% and 100% ethanol