Oct 11, 2021
Version 2
Coverslip Functionalization SOP003.v2.2 V.2
This protocol is a draft, published without a DOI.
- 1Arizona State University
- Human Cell Atlas Method Development Community
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,
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
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
Materials
Solution Preparation:
Silanization Solution
- 0.1 % (v/v) triethylamine (Millipore, TX1200)
- 0.2 % (v/v) allyltrichlorosilane (AllyltrichlorsilanSigma AldrichCatalog #107778 )
- dissolved in chloroform (ChloroformSigma AldrichCatalog #C2432-1L )
PDL (Poly-D-Lysine) Solution
- 0.1 Mass Percent Poly-D-Lysine (molecular weight 30,000-70,000 Da; Poly-D-lysine hydrobromide (PDL)Sigma AldrichCatalog #P7886 )
- Dissolved in nuclease-free water.
Additional Reagents:
- EthanolFisher ScientificCatalog #AC615090010
- ChloroformSigma AldrichCatalog #C2432-1L
- MethanolSigma AldrichCatalog #34860-1L-R
- Hydrochloric acidSigma – AldrichCatalog #320331-500ML
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
Part 1 - Clean coverslips
30m
30m
Wash for 00:30:00 via immersion in 1:1 mix of 37 % (v/v) HCl and methanol at Room temperature .
30m
Rinse 3x in DI H2O.
Fill beaker with Rnase-free water and autoclave coverslips to sterilize.
Rinse 1x in 70 % Ethanol .
Coverslips can be stored in 70 % EtoH indefinitely.
Part 2 - Silanize coverslips
Part 2 - Silanize coverslips
Dry at 60 °C .
Immerse in silanization solution in Fume-hood for 00:30:00 at Room temperature .
30m
Wash 1x with chloroform.
Wash 1x with ethanol.
Bake in a 60 °C oven for 02:00:00 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)
Part 3 - PDL coat coverslips (for cell culture on coverslips)
1h
1h
Immerse coverslips in PDL Solution for 01:00:00 at Room temperature .
1h
Rinse coverslips three times with nuclease-free water and then dry at Room temperature .
Silanized, PDL-coated coverslips may be stored at Room temperature in a desiccation chamber for weeks.