Jan 18, 2024
Single-Molecule Antibody Slides For Fluorescence Microscopy
- 1Univeristy of Cambridge
Protocol Citation: Rebecca Andrews 2024. Single-Molecule Antibody Slides For Fluorescence Microscopy . protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1qbbygr2/v1
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: January 18, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93745
Keywords: ASAPCRN
Abstract
This protocol describes how to create single-moelcule antibody slides for fluorescence microscopy.
Coverslip Cleaning
Coverslip Cleaning
1h 0m 10s
1h 0m 10s
Coverslips (24 x 50 mm, #1, VWR,Catalogue Number 48404-453) were argon plasma cleaned (Ar plasma cleaner, PDC-002, Harrick Plasma) for 00:30:00 .
30m
Surface preparation
Surface preparation
1h 0m 10s
1h 0m 10s
A trimmed gasket was placed on top of the slide (CultureWell‱ Reusable Gasket, 6mm diameter, Grace Bio-Labs, SKU: 103280).
Poly-L-Lysine 30 µL (0.01 % w/v PLL, P4707, Sigma-Aldrich) was placed in the wells for 00:30:00 .
30m
The PLL was removed, the wells washed three times with PBS (pH 7.4, 1x Gibco, Thermo Fisher Scientific, Catalogue number 10010023)
Depositing Antibodies
Depositing Antibodies
1h 0m 10s
1h 0m 10s
A secondary antibody of choice was added at a final concentration of 0.0002 mg/ml in PBS.
The antibodies were left in the wells for 5-10 seconds for sufficient surface density before the wells were washed three times with PBS.
10s
PBS 30 µL was left in the wells for imaging