Download In Situ Hybridization Protocols 2nd Edition (Methods in by Ian A. Darby (Editor) PDF

By Ian A. Darby (Editor)

Ian Darby updates the hugely profitable preferable version with an entire panoply of recent and enormously augmented state of the art suggestions. This necessary re-creation comprises easy and complex in situ hybridization suggestions - many now not lined within the first version - and comprises protocols for in situ hybridization of whole-mount embryo specimens, in situ hybridization on the electron microscope point, in situ detection of DNA fragmentation in apoptosis, localization of genes to specific chromosomes, and using DNA or RNA probes to notice expression in cells or tissue sections. ready by way of best investigators who've day-by-day labored with the suggestions, fine-tuning them for reliability, In Situ Hybridization Protocols, 2d variation, bargains a very up to date and prolonged selection of confirmed and without problems reproducible equipment appropriate for either the amateur and skilled investigator. Its cutting-edge protocols represent the hot average tools source within the box, person who will allow researchers effectively to reinforce and enhance their present experimental repertoire.

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Read or Download In Situ Hybridization Protocols 2nd Edition (Methods in Molecular Biology Vol 123) PDF

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2× SSC (before and after 4',6-diamidino-2-phenylindole [DAPI] staining wash). 3. Detection and Amplification 1. 1% Tween-20. 2. 1% Tween-20 in 4× SSC. Store at 4°C. 3. Avidin-FITC (fluorescein isothiocyanate, Vector): 500 µg/mL (stock solution). FITC detection working solution: 10 mL of avidin-FITC stock solution to 990 µL of detection mixture. Store in the dark at 4°C. Good for up to 6 mo. 4. Biotinylated goat anti-avidin antibody (Vector): 500 µg/mL (stock solution). Aliquots (50 µL each) can be kept at –20°C.

2. 5 min. 3. Quickly transfer the slides to a plastic jar with cold 70% ethanol for 2 min. Dehydrate the slides in 95% and 100% ethanol for 3 min each. Air dry and perform hybridization immediately. 4. Hybridization 1. Load 15 µL of denatured probe in hybridization solution onto each slide and cover with a 22 × 22 mm coverslip. Gently remove air bubbles and seal the edges with rubber cement to minimize evaporation. 2. Hybridize at 37°C in a humid chamber containing a water-soaked tissue. For repetitive probes, hybridize for a few hours or overnight.

Storage of Prepared Slides Mouse slides so stored have been used for RISH after 10 yr (10); however, human slides stored for long periods often do not band as well as comparatively new slides. 1. Label the slides with pencil on the frosted surface. 2. Pack in boxes without hinges (Kartell), with silica gel drying agent in a punctured plastic tube. 3. Seal the box with electrical tape and place at –20°C. 9. Exposure of Slides to Light To minimize random nicking of 5BrdU-substituted DNA, the slides should be protected from strong light whenever possible, at least until after hybridization.

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