|Materials Needed||Wash Solutions|
3 Slide Wash Chambers
10% SDS Centrifuge that can spin plates at 500 RPM
|Wash 1||Wash 2||Stock|
|10 ml||1 ml||20x SSC|
|0.5 ml||--||10% SDS|
|340 ml||350 ml||H2O|
Prepare the wash buffers, and have the wash chambers, slide racks, and drying centrifuge ready. The third glass chamber is left empty and used for carrying the slide rack from the last wash to the centrifuge.
Carefully remove the chamber from the heat source and dry it down with paper towels. Water between the sandwich halves can be wicked out with paper towels. If you have several chambers, process them a few at at time to get them into the first wash. Dry them off and remove the screws, but refrain from cracking open the covers until all chambers are ready to open. The time from opening the chamber, to the time the slide rack is immersed in the first wash should be minimized to prevent drying out of the probe. The slide chambers contain a small recess under one edge of the slide for prying the slide out of the chamber. Crack open the slide chamber and place the slide in a slide rack.
Place the slide rack into the first wash. Gently plunge the rack up and down. The trick is to get the cover slip to fall off without scratching the surface of the slide, or getting stuck on the slide rack. Some slide racks are constructed in such a way that it is very difficult to get the coverslip to fall free of the slide. Wheaton Slide racks are recommended because they contain no obstruction in the vicinity of the cover slip. Be careful not to let the surface of the slide dry at any time during the washes. If there are many slides to process, it is worth setting up two chambers containing Wash 1. Place a slide rack in the second chamber, and use the first chamber simply for emmersing the slides and shaking off the cover slip.
Once the cover slip is free, a few plunges up and down are suffucient to wash the slide. Move the rack to the second wash, and third wash with a few plunges each. The accumulated time of all three washes is usually less than five minutes. Even drying of the surface is important to avoid background problems. The centrifuge should be ready with a balance slide rack, and an empty rack padded with a paper towel. After the last wash, place the slide rack into the centrifuge and spin 5 minutes at 600 RPM.
Remove the slides and place them into a dust free box for scanning.
With the current scanning set-up there are two steps to quantifying the arrays. The first is scanning the slide to get a flourescence image. The second is gridding the scanned image to quantify the amount of flourescence at all the spots.