Dec 04, 2023
Version 2
Tetraspeck Bead Imaging V.2
- 1Yusuf Hamied Department of Chemistry, University of Cambridge
Protocol Citation: Joseph S Beckwith 2023. Tetraspeck Bead Imaging . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22br4l1y/v2Version created by Joseph S Beckwith
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 04, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 91759
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000509
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Abstract
Protocol for imaging tetraspeck beads on glass coverslips.
Slide Preparation
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).
30m
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.
5m
Add 50 μl of 0.01 % w/v poly-L-lysine (PLL, P4707, Sigma-Aldrich) to the well and wait for 10-20 min.
20m
Use a pipet to remove excess PLL.
2m
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.
5m
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.
3m
Remove excess solution using a pipet.
2m
Wash with 50 μl of filtered PBS. Pipet up and down in the corners of the well to wash. Repeat this step 3 times.
2m
Remove excess PBS and add 50 μl filtered PBS to the well. The sample should not dry out!
1m
Imaging
Imaging
Image the slide on a light microscope.