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    Single-Molecule Antibody Slides For Fluorescence Microscopy

    Rebecca Andrews

    Published: 2024-01-18 DOI: 10.17504/protocols.io.ewov1qbbygr2/v1
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    Abstract

    This protocol describes how to create single-moelcule antibody slides for fluorescence microscopy.

    Steps

    Coverslip Cleaning

    1.

    Coverslips (24 x 50 mm, #1, VWR,Catalogue Number 48404-453) were argon plasma cleaned (Ar plasma cleaner, PDC-002, Harrick Plasma) for 0h 30m 0s .

    Surface preparation

    2.

    A trimmed gasket was placed on top of the slide (CultureWellTM Reusable Gasket, 6mm diameter, Grace Bio-Labs, SKU: 103280).

    3.

    Poly-L-Lysine 30µL (0.01 % w/v PLL, P4707, Sigma-Aldrich) was placed in the wells for 0h 30m 0s.

    4.

    The PLL was removed, the wells washed three times with PBS (pH 7.4, 1x Gibco, Thermo Fisher Scientific, Catalogue number 10010023)

    Depositing Antibodies

    5.

    A secondary antibody of choice was added at a final concentration of 0.0002 mg/ml in PBS.

    6.

    The antibodies were left in the wells for 5-10 seconds for sufficient surface density before the wells were washed three times with PBS.

    7.

    PBS 30µL was left in the wells for imaging

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