Slide Subbing

Note: This protocol is from Jackson Bittencourt/Paul Sawchenko (1989), and is an abbreviated version of the lab protocol (from Simmons et al., 1989). My version was originally posted Oct. 3, 2012 and revised Feb. 12, 2013

1. Dump new slides into Acid Alcohol (70% EtOH + 1% HCl)
2. Hand wash* and transfer to distilled (DI)
3. Rack slides leaving one blank space at either end.
4. Soak the racked slides in DI + Alconox* for 30 min or more
5. Rinse out Alconox in DI for 1 - 2h, and leave soaking in DI overnight if possible
6. To preparing the subbing solution, use:

• 500 ml* DI in a 1000 ml beaker
• a funnel (preferably glass, but must be clean)
• ring stand
• filter paper
• 5.0 g gelatin (Knox Gelatin*, available at most grocery stores)
• 0.5 g Chromium Potassium Sulfate (On the shelf with the gelatin)

      5.  Heat water to 70 - 80º C (i.e., do not boil)

      6. Add gelatin and, after gelatin dissolves, add Chromium Potassium Sulfate

      7. Let cool to 40º - 60º C* and filter into glass Wheaton staining dish 

      8. Soak slides/racks for about 2 min each

      9. Put slide racks on tray, tipped to drain with frosting pointing down. Cover with

          lab diaper to protect from dust

    10. Move tray(s) to oven (37 - 40º C) overnight.

    11. Remove from oven & let cool to room temperature before putting away.

    12. Store slides in clean, dust free boxes. Wrap in plastic wrap for long term storage

*Notes:

1. New slides are often obviously dirty so the acid alcohol wash is an annoying but necessary step
2. Hand washing is a quick swipe front and back with a 4 x 4 gauze sponge, or similar cheap, sturdy, disposable and lint-free cloth.
3. Alconox. Use 7.5 g/L and make about 6 L (45 g) for 10 large racks. More soluble and effective in hot water. Recommend heating 3 L DI to 40 - 60º C, adding 45 g Alconox while stirring, then at to 3 L DI (RT), rather than using hot tap water
4. Subbing: 500 ml is barely enough to cover a rack in a large dish and gelatin is cheap so make plenty. Time can be save by subbing 2 racks in parallel. For 2 dishes make 1.5 L subbing sol'n.
5. Gelatin: The  Knox packs contain approx. 7.2 g gelatin,which means 3 packs are needed to make 1.5 L
6. Subbing works better with hot gelatin but is simply too hot to handle above 60º
7. Stored slides should be labelled as "Sub'd" and dated but last indefinitely if wrapped (i.e., protected from dust and moisture)

The secrets to subbing are keeping the slides and dishes clean, avoiding bubbles, and don't over do it. To keep the slides clean, 1) wear gloves (hands are oily, if not dirty),  2) avoid lint. Don't use paper towels for anything. Do use Kimwipes and other papers stated to be lint free. Set slides on the absorbent side of the lab diaper, but cover with the plastic side facing the slide, and 3) keep the slides either submerged or covered (even in the oven)  To keep the dishes clean, wash them immediately (because obviously dried gelatin doesn't come off glass easily). Because dust is the biggest enemy, the Wheaton dishes should be dried also and wrapped in plastic wrap for long-term storage. Use lint-free Kimwipes, not paper towels. Bubbles are a problem because they dry as raised rings of gelatin, potentially causing slides to stick together and always rendering the slides unusable. You always get bubbles when filling the Wheaton dish. Use a Kimwipe to remove bubbles by blotting them away or at least moving them to the sides of the dish and out of the way. When removing racks from the subbing solution, drain well and inspect each rack of slides making sure that there is no liquid remaining in the gaps between slides. 

Finally, figure out how many slides can be subbed and do that many or less. The best policy is to use entire boxes. Open, partial boxes should be discarded to avoid confusion and costly mistakes. Note that slides come 72/box and 20 boxes/case which means 1440 slides in a case. Currently we have enough racks to do about 15 boxes (> 1000 slides) in a run, but only one tray that fits the oven. The tray holds 8 large racks, which combined could take 384 slides but only 5 boxes (360 slides). Buy one more tray and the max run increases to 10 boxes, which is probably the practical capacity of the oven. The actual run should be limited to the number of slides that can be subbed without compromising on any of the steps in the protocol.

      

Large, 50-slide metal slide rack. Similar to 25-slide rack

Wheaton Staining dish to fit large slide rack