Apr 08, 2026
Immunostaining protocol for mitochondrial morphology and OXPHOS complex expression in iPSC-derived glutamatergic neurons
- Alfred Kibowen1,
- Kristen Brennand1,2,
- Kristen Brennand3
- 1Department of Psychiatry, Division of Molecular Psychiatry, Yale University School of Medicine, New Haven, CT 06511;
- 2Breakthrough Discoveries for thriving with Bipolar Disorder, Santa Monica, CA;
- 3Yale University School of Medicine
- Brennand Laboratory
Protocol Citation: Alfred Kibowen, Kristen Brennand, Kristen Brennand 2026. Immunostaining protocol for mitochondrial morphology and OXPHOS complex expression in iPSC-derived glutamatergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkzpkdl5r/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 07, 2026
Last Modified: April 08, 2026
Protocol Integer ID: 314601
Keywords: total oxphos antibody, expression of oxidative phosphorylation, protocol for mitochondrial morphology, oxidative phosphorylation, overall oxphos expression, expression levels of oxphos complex, oxphos complex expression, quantitative parameters of mitochondrial structure, mitochondrial morphology, mitochondrial structure, oxphos complex, oxpho, derived glutamatergic neuron, glutamatergic neuron, complexes in human ipsc, glutamatergic neurons this protocol, tomm20 antibody, antibody
Funders Acknowledgements:
BD2 Discovery
Abstract
This protocol describes the use of immunostaining to assess mitochondrial morphology and the expression of oxidative phosphorylation (OXPHOS) complexes in human iPSC-derived glutamatergic neurons (iGLUTs). Mitochondrial morphology is evaluated using a TOMM20 antibody, while overall OXPHOS expression is measured with a total OXPHOS antibody. Expected outcomes include quantitative parameters of mitochondrial structure (such as volume, area, sphericity, and network features) as well as the expression levels of OXPHOS complexes.
Materials
- 96-well imaging plate (PerkinElmer PhenoPlate 96-well, 6055300)
- Sucrose (S7903-250G)
- Paraformaldehyde (Thermofisher, 28908)
- Dulbecco’s Phosphate Buffered Saline (DPBS; Gibco, 14040133)
- Normal donkey serum (Jackson ImmunoResearch, 017-000-121)
- Triton (Thermofisher scientific, 85111)
- DAPI (Sigma, D9542)
- MAP2 antibody(Abcam, ab5392)
- TOMM20 antibody (Santa Cruz Biotechnology, sc-17764)
- Total OXPHOS antibody cocktail (Abcam, AB-317270)
- Donkey Anti-Mouse secondary antibody (Jackson ImmunoResearch, 715-605-151)
- Donkey Anti-Rabbit secondary antibody (Jackson ImmunoResearch, 711-165-152)
- Donkey Anti-Chicken secondary antibody (Jackson ImmunoResearch, 715-545-155)
- DragonFly Confocal Dual Spinning Disk microscope
Procedure
Seed 1.5 x 10^4 Day 5 iPSC-derived glutamatergic neurons (iGLUTs) into each well of a 96-well imaging plate and continue differentiation until Day 21 for antibody staining.
Prior to staining, fix cells with a fixative solution containing 4% paraformaldehyde and 4% sucrose in DPBS for 10 minutes at room temperature (RT).
After fixation, wash cells twice with DPBS and incubate the cells with blocking solution containing 2% normal donkey serum and 0.1% Triton in DPBS for 2 hours at RT.
Prepare primary antibodies in blocking solution with the following combinations and ratios:
for mitochondrial morphology: MAP2 (1:1000) + TOMM20 (1:200)
for OXPHOS complex expression: MAP2 (1:1000) + OXPHOS antibody cocktail (1:500)
Remove blocking solution and incubate the cells with primary antibodies at 4 °C overnight.
Prepare secondary antibodies in blocking solution at 1:500 dilution. The combination of secondary antibodies is determined by the species of the primary antibodies used in the experiment.
Wash cells with DPBS three times, then incubate the cells with secondary antibodies for 1 hour at RT.
After secondary antibody incubation, wash the cells three times with DPBS. During the second DPBS wash, add DAPI to DPBS (1 µL/ml) and incubate for 2 minutes at RT.
Image the plate using a DragonFly Confocal Dual Spinning Disk confocal microscope at 60x magnification and 1.4 numerical aperture objective.
After image acquisition, use the Surface module of Imaris 10.2 to quantify mitochondrial structure (volume, area, sphericity) and OXPHOS complex expression (fluorescent intensity).
To determine mitochondrial networking features, use previously published methods.
Protocol references
1. Chaudhry, A., Shi, R. 26 Luciani, D. S. A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells. Am J Physiol Endocrinol Metab 318, E87–E101 (2020).
2. Garcia, M. F. et al. Dynamic convergence of neurodevelopmental disorder risk genes across neurodevelopment. 2024.08.23.609190 Preprint at https://doi.org/10.1101/2024.08.23.609190 (2025).
3. Schrode, N. et al. Synergistic effects of common schizophrenia risk variants. Nat Genet 51, 1475–1485 (2019).
4. Michael Deans, P. J. et al. Functional implications of polygenic risk for schizophrenia in human neurons. Nat Commun 17, 1355 (2026).
Acknowledgements
Statistical analysis
- For each phenotype (e.g., mitochondrial sphericity), conduct a one-way ANOVA to compare groups, using Šidák’s correction for multiple comparisons.
Additional information
- For the data generated from this protocol:
- No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications.
- Data distribution was assumed to be normal, but this was not formally tested.
- Data collection was pseudo-randomized by randomizing the seeding of different groups in different areas of the 96-well plate.
- Data collection and analysis were not performed blind to the conditions of the experiments.
- No datapoints were excluded from the analyses.