Polylysine Coated Coverslip Preparation with SEM monolayer RBC prep

Materials Needed:
Clean, ½” #2 coverslips
Pasteur pipettes and bulbs 
Small Petri dishes or sample cups
Fine forceps
Coverslip holder for CPD
Super glue
SEM stubs
Silver paint
Rubber lifts

Chemicals Needed:
Polylysine
0.1M CAC buffer or P04 buffer
dd H2O
ETOH 30,50,70,90,100%

Equipment Needed:
Hood
Critical Point Dryer
Denton Sputter Coater

Note: If possible, make a duplicate coverslip for each sample.
Use 5ml cup for single, petri for duplicates

1. Clean coverslips with dd H2O and 95% ETOH, place them on rubber lifts

2. Place a drop of polylysine on each coverslip sufficient in size to cover it

3. Let rest with polylysine for 5-30 minutes

4. Remove excess polylysine by dipping coverslips in dd H2O

5. Dip in 0.1M buffer

6. Blot excess buffer and immediately apply coverslip with sample

7. Leave sample for 20 minutes

8. Transfer coverslips to 5ml cups filled with 0.1M buffer

9. Gently swirl, pipette excess sample to the original tube

10. Flood sample with dd H2O

11. Remove H2O and flood with ETOH (incrementally)
    a. Leave each solution of ETOH for 5 minutes (to allow continued removal of excess sample.)
    b. If using petri dishes be sure to move coverslips to prevent sticking

12. Add a second 100% ETOH change, prepare for CPD
    a. Place coverslip holder in small container with 100% ETOH
    b. Insert filler-coverslip … filler-weight (be sure to note orientation of sample)

13. Follow CPD instructions

14. Mount on labeled SEM stubs with a dot of super glue

15. Follow Sputter Coater instructions

16. Connect stub to top of coverslip with silver paint

17. Samples are now ready for SEM

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27 March 2004 DG