Apr 13, 2026

Immunofluorescent Image Acquisition and Quantification

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvoe447l4o/v1
  • Sabina Marciano1,
  • Roberta Marongiu2
  • 1Weill Cornell Medicine;
  • 2Weill Cornell Medical College
Jacquelyn Haytayan
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvoe447l4o/v1
Protocol Citation: Sabina Marciano, Roberta Marongiu 2026. Immunofluorescent Image Acquisition and Quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoe447l4o/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: April 13, 2026
Last Modified: April 13, 2026
Protocol  Integer ID: 314940
Keywords: ASAPCRN, rat dopaminergic n27 cell, cultivation of rat dopaminergic n27 cell, immunofluorescent image acquisition, automated quantification of immunofluorescent marker, immunofluorescent marker, cellprofiler, striatum, cellprofiler for object, intensity quantification, cell
Funders Acknowledgements:
Aligning Science Across Parkinson's
Collaboration Research Network
Abstract
This protocol describes the cultivation of rat dopaminergic N27 cells, transient transfection and immunoblottingThis protocol details the systematic acquisition and automated quantification of immunofluorescent markers (TH, mCherry, Iba1, and pSer129) in the Substantia Nigra (SN) and striatum. It utilizes a blinded workflow involving QuPath for regional delineation and CellProfiler for object and intensity quantification.
Materials
Materials & Software
  • Software:
QuPath: For digital pathology and ROI annotation.
ImageJ/Fiji: For ROI format conversion.
CellProfiler (v4.x/65): For high-throughput image analysis pipelines.
  • Markers: Tyrosine Hydroxylase (TH), mCherry, Iba1, and phospho-synuclein Ser129 (pSer129).

Equipment
  • Microscope: Olympus BX61TRF.
  • Camera: Hamamatsu Digital Camera C13440 ORCA-Flash 4.0.
  • Objective: 10x.
Image Acquisition
Set up the Olympus BX61TRF microscope with the 10x objective.
Acquire fluorescent images of nigral and striatal sections using the Hamamatsu ORCA-Flash 4.0 camera.
Maintain consistent exposure times and gain settings across all experimental groups to allow for intensity comparisons.
ROI Delineation (QuPath & ImageJ)
Import the raw 10x images into QuPath.
Manually delineate the Regions of Interest (ROIs) for the Substantia Nigra (SN) and striatum.
Export the defined ROIs into ImageJ to ensure compatibility with the downstream analysis pipeline.
Automated Quantification (CellProfiler)
Import images and corresponding ROIs into CellProfiler.
Blinding: Ensure investigators are blinded to the experimental conditions (genotype/treatment) before starting the analysis.
Run built-in-house pipelines to identify objects based on:
Cellular morphology
Fluorescence intensity parameters
name of built-in-house pipelines: Transd.eff.cpproj, TH_IBA1_PSYN_SN.cpproj, TH_IBA1_PSYN_STR.cpproj
Data Extraction
Total Cell Count: Quantify the number of objects (e.g., TH+ or Iba1+ cells).
Pixel Density/Intensity: Quantify marker intensity per area (mm2).
Data Normalization and Averaging
Calculate the cell counts or intensity values for each individual section.
Normalize counts to the area of the ROI (counts/mm2).
Average the values from all sections belonging to a single sample to generate a final representative value per subject.