Tetraspeck Bead Imaging
Joseph S Beckwith
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Abstract
Protocol for imaging tetraspeck beads on glass coverslips.
Steps
Slide Preparation
Glass coverslips (Fisher Scientific, 12373128, #1 thickness 22 mm x 50 mm) were plasma cleaned for 30 min (Ar plasma cleaner, PDC-002, Harrick Plasma).
Stick a frame-seal slide chamber (9 mm x 9 mm, SLF0201, Biorad) on the cover glass. Use some blunt tweezers to press down the sticker on the glass.
Add 50 μl of 0.01 % w/v poly-L-lysine (PLL, P4707, Sigma-Aldrich) to the well and wait for 10-20 min.
Use a pipet to remove excess PLL.
Wash with 50 μl of filtered (0.02 μm syringe filter, Whatman, 6809-1102) PBS. Pipet up and down in the corners of the well to wash. Repeat this step 3 times.
Remove excess PBS and add 50 μl of the diluted TetraSpeck (1:625 dilution, 0.1 μm diameter TetraSpeck Microspheres, Thermo Fisher) beads to the well. Wait 2-3 minutes to let the beads settle and attach to the PLL-coated glass.
Remove excess solution using a pipet.
Wash with 50 μl of filtered PBS. Pipet up and down in the corners of the well to wash. Repeat this step 3 times.
Remove excess PBS and add 50 μl filtered PBS to the well. The sample should not dry out!
Imaging
Image the slide on a light microscope.
