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Contents

Contents

• 1. Preparation of microscope slides-silane coating
• Summary
• Reagents
• Equipment

• 2 Flow chart :
• 3 Fixation, processing, sectioning
• 3.1 Fixation and embedding
• 3.1.1 Fixatives and fixation
• 3.1.2 Effect of fixative and fixation method
• 3.1.3 Embedding
• 3.2 Fixed, paraffin-embedded tissue
• Summary
• 3.2.1 Perfusion-fixation
• 3.2.2 Immersion-fixation
• 3.2.3 Paraffin wax infiltration
• 3.2.4 Embedding
• 3.2.5 Sectioning
• 3.2.6 Storage
• 3.3 Fixed-frozen tissue
• 3.3.1 Perfusion / immersion-fixation and sucrose infiltration
• 3.3.2 Embedding
• 3.3.3 Sectioning
• 3.3.4 Storage
• 3.4 Fresh-frozen tissue
• 3.4.1 Harvesting and embedding tissue
• 3.4.2 Sectioning and fixation
• 3.4.3 Storage
• 4 Pre-hybridisation tissue processing
• 4.1 Effect of pre-hybridisation treatments
• 4.2 Fixed, paraffin-embedded tissue
• 4.2.1 De-waxing, re-hydration, proteinase K digestion
• 4.2.2 Acetylation
• 4.2.3 Storage
• 4.3 Fixed-frozen tissue
• 4.4 Fresh-frozen tissue
• 5 In vitro transcription
• 5.1 Synthesis of single-stranded RNA probes
• 5.1.1 In vitro transcription
• 5.1.2 Design of cRNA probes for ISH
• 5.1.3 Choice of radio-label : effect on S/N in ISH
• 5.1.4 Alkaline hydrolysis
• 5.2 Preparation of template DNA for in vitro transcription
• 5.3 Synthesis of cRNA probes for in situ hybridisation
• 5.4 Characterisation and purification of cRNA probes
• 5.4.1 Denaturing polyacrylamide gel electrophoresis
• 6 Flow chart
• 7 Hybridisation and post-hybridisationwashes
• 7.1 Hybridisation and post-hybridisation washes
• 7.1.1 Effect of hybridisation temperature on S/N in ISH
• 7.1.2 Effect of hybridisation buffer and probe concentration
• 7.2 Hybridisation
• 7.2.1 Control tissues and probes
• 7.2.2 Hybridisation
• 7.3 Post-hybridisation washes
• 8 Autoradiography
• 8.1 X-ray film autoradiography
• 8.2 Liquid film emulsion autoradiography
• 8.2.1 Choice of emulsion
• 8.2.2 Storage of emulsion and preparation of darkroom
• 8.2.3 Slide dipping and autoradiograph exposure
• 9 Analysis of slide autoradiographs
• 9.1 Dark field / bright field and epi-illuminationmicroscopy
• 9.2 Common problems
• 10 Miscellaneous
• 10.1 Advantages and disadvantages
• 10.2 ISH on mammalian embryos
• Footnotes

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