Jul 28, 2023
immunofluorescent staining with anti-GFP and anti-CD63 antibodies
- 1KU Leuven
Protocol Citation: rosanne.wouters, Peter Vangheluwe 2023. immunofluorescent staining with anti-GFP and anti-CD63 antibodies. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2pypg5d/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: June 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82751
Keywords: ASAPCRN
Funders Acknowledgements:
ASAP
Abstract
This protocol was used for immunofluorescent staining in fixed HeLa cells with anti-GFP and anti-CD63 antibodies, followed by confocal imaging.
Protocol materials
FluorSave™ ReagentMerck MilliporeSigma (Sigma-Aldrich)Catalog #345789
plate cells on glass coverslips and grown until they reach 60% conlfuency
fix cells with 4% paraformaldehyde for 20 min at room temperature
permeabilize cells with 0.1% Triton X-100 in PBS for 5 min
block for 1 h with blocking buffer (PBS with 0.5% Tween20, 0.1% BSA, 0.2% FBS)
incubate coverslips with primary antibodies for 2 h at room temperature
(anti-CD63, exbio, 11-343-C100, mouse; anti-GFP, abcam, ab13970, chicken)
wash coverslips 3 times with PBS-T
incubated coverslips 30 min with secondary antibodies
(goat-anti-mouse-AlexaFluor647, goat-anti-chicken-AlexaFluor488)
wash coverslips 3 times with PBS-T
incubate coverslips with DAPI
wash coverslips 3 times with PBS-T
mounte coverslips usingFluorSave™ ReagentMerck MilliporeSigma (Sigma-Aldrich)Catalog #345789
images were acquired using an LSM780 con- focal microscope (Zeiss) with a 10x or 40× objective
colocalization analysis was performed with Fiji plugin Jacop