Apr 28, 2026
Immunocytochemistry, Confocal Imaging, and Quantitative Analysis of iNeurons
- Felix Kraus,
- Harper JW1
- 1harvard university
Protocol Citation: Felix Kraus, Harper JW 2026. Immunocytochemistry, Confocal Imaging, and Quantitative Analysis of iNeurons. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6jyj1vqe/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: March 16, 2026
Last Modified: April 28, 2026
Protocol Integer ID: 313300
Keywords: ASAPCRN, disk confocal imaging of induced neuron, quantitative analysis of ineuron, quantification of synaptic protein puncta, confocal imaging, confocal microscope, synaptic protein puncta, synaptic marker, ineuron, immunocytochemical staining, w1 spinning disk confocal microscope, identification of tyrosine hydroxylase, antibody labeling, induced neuron, tyrosine hydroxylase, neuron, fluorescence
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: 024268, 025160
Abstract
This protocol describes fixation, immunocytochemical staining, and spinning disk confocal imaging of induced neurons (iNeurons) at defined stages of differentiation (see dx.doi.org/10.17504/protocols.io.br9em93e for a typical differentiation protocol). The workflow includes antibody labeling of neuronal and synaptic markers followed by imaging using a Yokogawa CSU-W1 spinning disk confocal microscope. Downstream quantitative analysis pipelines are provided for the identification of tyrosine hydroxylase (TH)–positive neurons and for the quantification of synaptic protein puncta using automated segmentation and fluorescence analysis in Python.
Guidelines
- Maintain consistent laser intensity and exposure times across all samples to allow quantitative comparisons.
- All washes should be performed gently to avoid detaching neurons.
- Protect fluorescent samples from light whenever possible.
- Prepare all buffers fresh or verify that stored buffers are free of contamination.
- Image acquisition parameters should remain constant within experiments intended for quantitative analysis.
**Image Acquisition Guidelines**
- Maintain identical exposure times and laser intensities for all samples.
- Acquire images using sequential channel acquisition to avoid spectral bleed-through.
- Export images in ND2 format for downstream computational analysis.
**Post-processing**
- Brightness and contrast adjustments are performed in ImageJ/FIJI using identical minimum and maximum display values across datasets.
Materials
**Reagents**
- 4% Paraformaldehyde in PBS (Electron Microscopy Sciences, #15710, EM grade)
- PBS (1×)
- Triton X-100
- Tween-20
- Bovine Serum Albumin (BSA)
- Hoechst 33342
**Primary antibodies**
- anti-MAP2 (Cell Signaling Technology, 4542S, 1:500 dilution)
- anti-TH (Sigma, MAB318, 1:500)
- anti-SYN1(Abcam, ab254349, 1:500 dilution)
- anti-tubulin (betaIII) (Abcam, ab41489, 1:500 dilution)
- anti-NEFH (Abcam, ab4680, 1:500 dilution)
- anti-Bassoon (BSN) (Enzo ADI-VAM-PS003-D, 1:500 dilution)
- anti-Synaptophysin (SYP) (Abcam, ab32127, 1:500 dilution)
**Secondary antibodies**
- Thermo Scientific fluorescent secondary antibodies (1:250)
**Equipment**
- Yokogawa CSU-W1 spinning disk confocal
- Nikon Eclipse Ti2-E motorized microscope
- Nikon objectives:
- Plan Apochromat 40×/0.40 NA air
- Plan Apochromat 60×/1.42 NA oil
- Plan Apochromat 100×/1.45 NA oil
- Nikon LUN-F XL solid-state laser combiner
- Hamamatsu ORCA-Fusion BT CMOS camera
- NIS-Elements acquisition software
- ImageJ/FIJI software
- Python environment for automated image analysis
- R environment for downstream statistical analysis
Safety warnings
⚠️ Paraformaldehyde (PFA) is toxic and should be handled in a chemical fume hood with appropriate PPE.
⚠️ Triton X-100 is an irritant; avoid inhalation and skin contact.
⚠️ Dispose of chemical waste according to institutional biosafety guidelines.
Before start
1. Pre-warm 4% paraformaldehyde (PFA) in PBS to 37 °C.
2. Prepare the following buffers:
- PBST**: PBS + 0.02% Tween-20
- Permeabilization buffer**: PBS + 0.5% Triton X-100
- Blocking buffer**: 3% BSA in PBS
3. Prepare primary and secondary antibody dilutions in 3% BSA-PBS**.
4. Ensure that the confocal microscope and lasers are properly calibrated.
Before Start
Pre-warm 4% paraformaldehyde (PFA) in PBS to 37 °C.
Prepare the following buffers:
**PBST**: PBS + 0.02% Tween-20
**Permeabilization buffer**: PBS + 0.5% Triton X-100
**Blocking buffer**: 3% BSA in PBS
Prepare primary and secondary antibody dilutions in 3% BSA-PBS**.
Ensure that the confocal microscope and lasers are properly calibrated.
Procedure
Remove culture medium from differentiated iNeurons.
Add 4% PFA in PBS (pre-warmed to 37 °C).
Incubate cells for 30 minutes at 37 °C.
Remove PFA and rinse cells once with PBS.
Incubate cells in 0.5% Triton X-100 in PBS for 15 minutes at room temperature.
Wash cells three times with PBST (0.02% Tween-20 in PBS).
Incubate cells in 3% BSA in PBS for 10 minutes at room temperature.
Wash cells three times with PBST.
Dilute primary antibodies in 3% BSA-PBS.
Add antibody solution to cells.
Incubate for 3 hours at room temperature.
Wash cells three times with PBST.
Dilute secondary antibodies 1:400 in 3% BSA-PBS.
Incubate cells with secondary antibodies for 1 hour at room temperature.
Add Hoechst 33342 (1:10,000 in PBST).
Incubate for 5 minutes.
Wash cells three times with PBST.
Cells are now ready for imaging.
Confocal Imaging
Images are acquired at room temperature using a Yokogawa CSU-W1 spinning disk confocal system mounted on a Nikon Eclipse Ti2-E microscope.
Lasers are applied sequentially using a Nikon LUN-F XL laser combiner.
Camera: Hamamatsu ORCA-Fusion BT CMOS (16-bit acquisition).
Laser excitation settings:
405 nm: 80 mW
488 nm: 80 mW
561 nm: 65 mW
640 nm: 60 mW
Dichroic mirror: Semrock Di01-T405/488/568/647
Emission filters:
405 nm: Chroma ET455/50m
488 nm: Chroma ET525/50m
561 nm: Chroma ET605/52m
640 nm: Chroma ET705/72m
Software: NIS-Elements
Data Analysis
Time-lapse calcium imaging datasets are analyzed using a custom Python pipeline_.
ND2 stacks are read using the nd2 Python package_.
Maximum intensity projections are generated.
Segmentation is performed using CellposeSAM with a pretrained model optimized for neuronal soma.
Segmentation masks are filtered by area (100–10,000 pixels).
For each valid region:
Centroid coordinates are extracted.
Mean fluorescence intensity is calculated across all frames.
Output files include:
16-bit TIFF segmentation masks
Visualization overlays
CSV files containing centroid coordinates and fluorescence traces
Synaptic puncta from markers such as Synaptophysin (SYP) and Bassoon (BSN) are quantified using a custom Python pipeline_.
Maximum-intensity projection images are parsed for experimental metadata.
Cytoskeletal channels generate neuronal masks.
Background subtraction and segmentation detect synaptic puncta.
Object morphology and fluorescence intensity are measured.
Object-level colocalization analysis is performed between SYP and Bassoon puncta.
Output files include:
Cleaned processed images
QC overlays
CSV files containing per-object and per-image measurements
Downstream statistical analysis and visualization are performed in R_.
Expected Results
Successful staining should reveal:
MAP2-positive neuronal morphology
TH-positive dopaminergic neurons where applicable
Synaptic puncta labeled by SYP and Bassoon
Quantitative outputs include:
Per-cell fluorescence measurements
Synaptic puncta counts and intensities
Colocalization metrics
Troubleshooting
Weak signal
Antibody concentration too low
Increase antibody concentration or incubation time
High background
Insufficient washing
Increase wash duration or PBST concentration
Cell loss
Harsh pipetting
Perform gentle buffer exchanges
Segmentation errors
Poor image contrast
Adjust acquisition settings or preprocessing parameters