Aug 02, 2023
Fluorescence intensity analyses
- Ayse Ulusoy1,
- Sinead O'Sullivan1,
- Michael Helwig1,
- Angela Rollar1,
- Shirley Lee1,
- Michael Klinkenberg1,
- Rita Pinto-Costa1,
- Donato Di Monte1
- 1DZNE
Protocol Citation: Ayse Ulusoy, Sinead O'Sullivan, Michael Helwig, Angela Rollar, Shirley Lee, Michael Klinkenberg, Rita Pinto-Costa, Donato Di Monte 2023. Fluorescence intensity analyses. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8jrmdg2w/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: August 25, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69176
Keywords: ASAPCRN
Abstract
Fluorescence intensity analyses
Collect confocal z-stack images from fluorescent-labeled tissue samples. Here we used DHE-treated mice tissue counter-stained with human alpha-synuclein or tissue co-immunolabeled with a human alpha-synuclein and SynO2 antibodies.
Ox-DHE fluorescent signal measurement
Ox-DHE fluorescent signal measurement
Create a 3D surface rendering model of h-alpha-synuclein–immunoreactive
DMnX neurons using the Imaris software.
By applying a constant intensity threshold select ox-DHE puncta and filter through h-alpha-synuclein–immunoreactive neuronal surfaces. This will allow for specific detection of ox-DHE puncta within immunoreactive
neurons
Quantify puncta on a per-cell basis using the quantification tools.
Syn-O2 fluorescence intensity measurement
Syn-O2 fluorescence intensity measurement
Generate 2D images that from 5 undetermined -thick z-stack images using maximum
intensity projection function of the Zen software (Carl Zeiss).
Select human alpha-synuclein labeled neurons by applying a 120µm2
size exclusion filter
Measure Syn-O2 intensity within these cells using the measure tool.