Oct 11, 2021

Public workspaceCoverslip Functionalization SOP003.v2.2 V.2

This protocol is a draft, published without a DOI.
Coverslip Functionalization SOP003.v2.2
  • Rory Kruithoff1,
  • Douglas Shepherd1
  • 1Arizona State University
  • Human Cell Atlas Method Development Community
Rory Kruithoff
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Protocol Citation: Rory Kruithoff, Douglas Shepherd 2021. Coverslip Functionalization SOP003.v2.2. protocols.io https://protocols.io/view/coverslip-functionalization-sop003-v2-2-byxxpxpnVersion created by Rory Kruithoff
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: October 11, 2021
Last Modified: October 11, 2021
Protocol Integer ID: 53975
Keywords: Coverslip, silanization, poly-d-lysine, coverslip cleaning, tissue adhesion, cell adhesion, allyltrichlorosilane, coverslip surface for improved adhesion, silanized coverslip, improved adhesion, pdl coat coverslip, coverslip functionalization, covalent adhesion of polyacrylamide gel, clean coverslip, adhesion, coverslip surface, covalent adhesion, coverslip, polyacrylamide gel, protein anchoring system, coating, embedded tissue, cells to the surface, gel, polyacrylamide, surface, tissue,
Abstract
Document Summary:
This document, SOP003 – Coverslip Functionalization, describes the process for cleaning and modifying the coverslip surface for improved adhesion of a sample.This procedure describes two methods of functionalization, silanization and poly-d-lysine (PDL) coating. Silanization is a method used to promote covalent adhesion of polyacrylamide gel-embedded tissue while poly-d-lysine (PDL) coating of silanized coverslips provides a protein anchoring system which improves adhesion of living cells to the surface of the coverslip.

Quick Overview:
Part 1 - Clean coverslips
Part 2 - Silanize coverslips
Part 3 - PDL coat coverslips
Attachments
Icon for Coverslip Functionalization SOP003.v2.2.pdf
Coverslip Functional...
214KB
Guidelines
v2.2 revision notes
-Added Bottiger lab and OsmFISH references.


References:
Moffitt, J. R., Hao, J., Bambah-Mukku, D., Lu, T., Dulac, C., & Zhuang, X. (2016). High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing. Proceedings of the National Academy of Sciences, 113(50), 14456-14461. https://doi.org/10.1073/pnas.1617699113

Boettiger Lab Github: https://github.com/BoettigerLab/protocols/blob/master/MatrixEmbedding.md

OsmFISH dx.doi.org/10.17504/protocols.io.psednbe
Materials
Solution Preparation:

Silanization Solution
  • Concentration0.1 % (v/v) triethylamine (Millipore, TX1200)
  • Concentration0.2 % (v/v) allyltrichlorosilane (ReagentAllyltrichlorsilanMerck MilliporeSigma (Sigma-Aldrich)Catalog #107778 )
  • dissolved in chloroform (ReagentChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #C2432-1L )

PDL (Poly-D-Lysine) Solution
  • Concentration0.1 Mass Percent Poly-D-Lysine (molecular weight 30,000-70,000 Da; ReagentPoly-D-lysine hydrobromide (PDL)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7886 )
  • Dissolved in nuclease-free water.

Additional Reagents:
  • ReagentEthanolFisher ScientificCatalog #AC615090010
  • ReagentChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #C2432-1L
  • ReagentMethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #34860-1L-R
  • Reagent Hydrochloric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #320331-500ML
Troubleshooting
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Using 40-mm-diameter #1.5 coverslips
Part 1 - Clean coverslips
30m
Wash for Duration00:30:00 via immersion in 1:1 mix of Concentration37 % (v/v) HCl and methanol at TemperatureRoom temperature .
30m
Wash
Rinse 3x in DI H2O.
Wash
Fill beaker with Rnase-free water and autoclave coverslips to sterilize.
Rinse 1x in Concentration70 % Ethanol .
Wash
Coverslips can be stored in Concentration70 % EtoH indefinitely.
Part 2 - Silanize coverslips
Dry at Temperature60 °C .
Immerse in silanization solution in Fume-hood for Duration00:30:00 at TemperatureRoom temperature .
30m
Wash 1x with chloroform.
Wash
Wash 1x with ethanol.
Wash
Bake in a Temperature60 °C oven for Duration02:00:00 to dehydrate the silane layer.
2h
Remove from oven and let coverslips cool.
Store in a desiccated chamber for weeks.
Part 3 - PDL coat coverslips (for cell culture on coverslips)
1h
Immerse coverslips in PDL Solution for Duration01:00:00 at TemperatureRoom temperature .
1h
Rinse coverslips three times with nuclease-free water and then dry at TemperatureRoom temperature .
Wash
Silanized, PDL-coated coverslips may be stored at TemperatureRoom temperature in a desiccation chamber for weeks.