Nov 11, 2023
Immunofluorescence and object-based colocalization analysis
- 1University of California, San Diego
Protocol Citation: Leonardo A Parra-Rivas 2023. Immunofluorescence and object-based colocalization analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5xdodg1b/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: November 11, 2023
Last Modified: November 11, 2023
Protocol Integer ID: 90805
Abstract
Immunofluorescence and object-based colocalization analysis
Immunofluorescence
Immunofluorescence
For immunostaining, DIV17-21 neurons were fixed in 100% ice-cold methanol solution
for 15 min at room temperature, followed by extraction in 0.1 % Triton X-100
for 10 min, blocked in 10% BSA for 1 h at room temperature, and incubated
overnight at 4°C
with primary antibodies. We found that the methanol fixation was better than
paraformaldehyde fixation for pSer129 α-syn staining.
After washing with 1x PBS, neurons were blocked again for 30 min at room temperature and incubated with secondary antibodies (Alexa Fluor antibodies from Invitrogen
(RRID:AB_2633275, RRID:AB_2762824,RRID:AB_2633282) (1:500)
for 1 h at room temperature.
Images were acquired at 40X magnification. Z-stack
images were obtained as previously described , and all images were acquired and
processed using the MetaMorph
Microscopy Automation and Image Analysis Software (RRID:SCR_002368)(https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#gref).
Object-based colocalization analysis
Object-based colocalization analysis
For α-syn Ser129 and VAMP2 colocalization analysis in cultured neurons, 5-6 regions
of interest (ROIs) of 200x200 pixels were placed on each image. A total of 26
ROIs were used for this analysis.
Object-based colocalization analysis was done
using MATLAB (RRID:SCR_001622)( http://www.mathworks.com/products/matlab/). First, automatic puncta detection was done using local maxima. Next, detected puncta are said to have co-localized if the distance between their centers is less than the maximum radius of the two
particles.