Poly-L-Lysine Covered Slides Preparation (Triton Pre-Cleaning)

Poly-l-lysine Covered Slides Preparation (Triton pre-cleaning)

Last updated: July 07, 2003

Materials Qty Ordering info

Glass microscope slides 240 Corning #2947

Slide rack 8 Shandon Lipshaw #121 (800-245-6212) for 30 slides

Slide chamber 9 Shandon Lipshaw #121

Plastic or glass beaker3 5 L volume

Magnets3

ddH2O ~30 L

DI-H2Ofor rinsing of glass chambers

Triton X-10028 mLBio-Rad 161-0407

NaOH 350 g

95% Ethanol 1890 mL

Poly-L-lysine 315 mL Sigma P-8920

Tissue culture PBS 1X315 mL

Vacuum oven (45C)

Slide box (plastic only)3for 100 slides

1. Before you start to prepare slides, check each original piece of glass (Primary quality is very important.)

2. Place slides in slide racks. Place racks in chambers.

3. Prepare 1%TRITON PRE-CLEANING SOLUTION:

Dissolve 28mL BIO-RAD TRITON X-100 in 2772 mL ddH2O.

Total volume is 2800 mL; stir until completely mixed.

4. Pour solution into chambers with slides; cover chambers with lids. Mix on orbital shaker for 1-2 hr.

5. Fill fresh chamber with ddH2O. Transfer rack to fresh chamber. Rinse vigorously by plunging rack up and down for 1/2 min. Rinse original chamber with DI-H2O.

Repeat rinses in original chamber 5X with fresh ddH2O (1/2 min each time). Remove all traces of detergent. Leave rack in chamber in ddH2O.

6. Repeat whole procedure for each of 8 racks , using an additional chamber for the initial rinsing.

7. Prepare CLEANING SOLUTION:

Dissolve completely 350 g NaOH in 1260 mL ddH2O.

Add 1890 mL 95% ethanol. Total volume is 3500 mL; stir until completely mixed.

8. Pour solution into chambers with slides; cover chambers with lids. Mix on an orbital shaker for 3 hr. Once slides are clean, they should be exposed to air as little as possible. Dust particles will interfere with coating and printing.

9. Fill fresh chamber with ddH2O. Quickly transfer a rack to fresh chamber. Rinse vigorously by plunging rack up and down for 1/2 min. Rinse original chamber with DI-H2O. (It is critical to remove all traces of NaOH-ethanol.)

Repeat rinses in original chamber 5X with fresh ddH2O (1/2 min each time). Remove all traces of NaOH-ethanol. Leave rack in chamber in ddH2O.

10. Repeat whole procedure for each of 8 racks, using an additional chamber for initial rinsing.

11. Prepare POLY-L-LYSINE SOLUTION :

Stir 315 mL tissue culture PBS with 2520 mL ddH2O.

Add 315 mL poly-L-lysine. Total volume is 3150 mL. Stir.

Use plastic graduated cylinder and beaker.

12. Replace ddH2O from each chamber with rack with poly-L-lysine solution. Put on orbital shaker for 40 min.

13. Prepare for the next step by placing paper towels on 4 centrifuge plate carriers (to absorb liquid). Transfer a rack to the fresh chamber filled with ddH2O. Plunge carefully up and down 5X to rinse. Place the rack on a centrifuge plate carrier. Repeat rinsing for 3 more racks. It is very important to do it as quickly as possible! Spin 5 min. at 750 rpm.

Transfer slide racks to tray for transport to the vacuum/non-vacuum oven.

14. Dry slide racks in 45C vacuum oven (or 65C non-vacuum oven) for 10 min. (Vacuum is optional).

15. Meanwhile, start to repeat procedure with next 4 racks. Be sure that the end of the spin cycle coincides with the end of the previous drying cycle in an oven.

16. Store slides in a closed slide box (plastic only, without rubber or foam or cork mat bottom).

17. BEFORE PRINTING ARRAYS:

Check that polylysine coating is not opaque.

Test print, hyb and scan sample slides to determine slide batch quality.