Chapter 10
Hybridization Methods (Southern and Northern Blotting)
KAI-OLAF NETZER . . . • . . . . . . . . . • • . . . . . • . . . . . . . . . . . • . . • . . . 126
Subprotocol 1: Preparation of Dot Blots for DNA ............... 129
Subprotocol 2: Preparation of Southern Blots for Genomic DNA ... 131
Subprotocol 3: Hybridization of DNA Blots .................... 135
Subprotocol 4: Preparation of RNA Dot Blots .................. 139
Subprotocol 5: Preparation of Northern Blots .................. 140
Subprotocol 6: Hybridization of RNA Blots .................... 143
Subprotocol 7: Membrane Stripping ......................... 147
Chapter 11
Specimen Preparation Techniques for Cytogenetic Analysis
by Fluorescence in situ Hybridization
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN
AND PETER LICHTER . • . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • • . 148
Subprotocol 1: Cultivation of Blood Lymphocytes ............... 149
Subprotocol 2: Preparation of Metaphase Chromosomes
and Interphase Nuclei from Blood Lymphocytes .... 151
Subprotocol 3: Pepsin Digestion of Blood Lymphocytes .......... 153
Subprotocol 4: Preparation of Fibroblasts for FISH
(Adherently Growing Cells) .................... 154
Subprotocol 5: Treatment of Blood Smears .................... 155
Subprotocol 6: Bone Marrow Smears ......................... 156
Subprotocol 7: Clinical Material Obtained from Solid Tumors:
Pretreatment of Slides with Poly-L-Lysine ......... 157
Subprotocol 8: Preparation of Single Cell Suspensions
from Solid Tumor Material .................... 158
Subprotocol 9: Preparation of Prozen Sections ................. 160
Subprotocol 10: Paraffin-Embedded Tissue Sections ............. 161
Chapter 12
Chromosome Analysis by Fluorescence in situ Hybridization
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER •....•.•..... 166
Subprotocol1: Specimen Preparation ........................ 170
Subprotocol 2: Probe LabeHing by Nick Translation ............. 171
Subprotocol 3: Dot-Blot Assay to Test LabeHing ................ 174
Subprotocol 4: Probe Mixture and Denaturation for Fluorescence
in situ Hybridization ......................... 175
Contents
IX
Hybridization Methods (Southern and Northern Blotting)
KAI-OLAF NETZER . . . • . . . . . . . . . • • . . . . . • . . . . . . . . . . . • . . • . . . 126
Subprotocol 1: Preparation of Dot Blots for DNA ............... 129
Subprotocol 2: Preparation of Southern Blots for Genomic DNA ... 131
Subprotocol 3: Hybridization of DNA Blots .................... 135
Subprotocol 4: Preparation of RNA Dot Blots .................. 139
Subprotocol 5: Preparation of Northern Blots .................. 140
Subprotocol 6: Hybridization of RNA Blots .................... 143
Subprotocol 7: Membrane Stripping ......................... 147
Chapter 11
Specimen Preparation Techniques for Cytogenetic Analysis
by Fluorescence in situ Hybridization
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN
AND PETER LICHTER . • . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • • . 148
Subprotocol 1: Cultivation of Blood Lymphocytes ............... 149
Subprotocol 2: Preparation of Metaphase Chromosomes
and Interphase Nuclei from Blood Lymphocytes .... 151
Subprotocol 3: Pepsin Digestion of Blood Lymphocytes .......... 153
Subprotocol 4: Preparation of Fibroblasts for FISH
(Adherently Growing Cells) .................... 154
Subprotocol 5: Treatment of Blood Smears .................... 155
Subprotocol 6: Bone Marrow Smears ......................... 156
Subprotocol 7: Clinical Material Obtained from Solid Tumors:
Pretreatment of Slides with Poly-L-Lysine ......... 157
Subprotocol 8: Preparation of Single Cell Suspensions
from Solid Tumor Material .................... 158
Subprotocol 9: Preparation of Prozen Sections ................. 160
Subprotocol 10: Paraffin-Embedded Tissue Sections ............. 161
Chapter 12
Chromosome Analysis by Fluorescence in situ Hybridization
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER •....•.•..... 166
Subprotocol1: Specimen Preparation ........................ 170
Subprotocol 2: Probe LabeHing by Nick Translation ............. 171
Subprotocol 3: Dot-Blot Assay to Test LabeHing ................ 174
Subprotocol 4: Probe Mixture and Denaturation for Fluorescence
in situ Hybridization ......................... 175
Contents
IX