Feb 25, 2025
Immunolabeling of endogenous proteins for fluorescence microscopy in cultured mammalian cells
- Ahmad Shami1,
- Hera Saqub1,
- Samantha C Lewis1
- 1UC Berkeley
Protocol Citation: Ahmad Shami, Hera Saqub, Samantha C Lewis 2025. Immunolabeling of endogenous proteins for fluorescence microscopy in cultured mammalian cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldr1xxg5b/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: February 18, 2025
Last Modified: February 25, 2025
Protocol Integer ID: 120859
Keywords: Immunofluorescence, Mitochondrial protein detection, mitochondria, fluorescence microscopy
Funders Acknowledgements:
National Institutes of Health
Grant ID: R35GM147218
Abstract
This protocol details the detection of endogenous proteins by immunofluorescence. This is our standard protocol for imaging endogenous proteins associated with mitochondria and other membrane-bound organelles.
Guidelines
Introduction
This protocol provides detailed instructions for detection of endogenous proteins in fixed cells by immunofluorescence (Immunofluorescence Assay/IFA). The protocol results in a fluorescent sample, to be visualized using a high-resolution fluorescence confocal microscope. Target proteins appear as distinct signals corresponding to the fluorophore-conjugated secondary antibodies, allowing the spatial analysis of subcellular structures, protein interactions, and nucleic acids.
Materials
- Proliferating cancer cell line or fibroblasts at 40% confluency
- 1X Phosphate-buffered saline (PBS), pH 7.4
- Fixative: 4% Paraformaldehyde in PBS, pH 7.4
- Quenching Solution: 1M Glycine in PBS, pH 7.4
- Permeabilization Solution: 0.1% Triton-X 100 in PBS, pH 7.4
- Blocking Solution: 0.1% Tween in 1X TBS + 1% BSA (Bovine Serum Albumin)
- Glass bottom poly-D-lysine-coated dishes (P35GC-1.5-14-C, MatTek)
- Primary antibody (specific to the target protein)
- Secondary antibody conjugated to fluorophore of choice
Method
Method
4d 1h 1m
4d 1h 1m
Prepare the sample by plating cells in 2 mL of media at a 20% density in a glass bottom poly-D-lysine-coated dish (P35GC-1.5-14-C, MatTek).
Grow cells for 24:00:00 until they reach a confluency of approximately 40%.
1d
Aspirate media and fix the cells with 2 mL of pre-warmed 4% PFA in PBS.
Incubate for 00:20:00 at Room temperature on a shaker gently rotating.
20m
Quench the reaction by adding 200 µL of 1M glycine in PBS to the fixed sample and incubate for 00:01:00 at Room temperature .
1m
Aspirate the quenched fixative and wash the sample once with PBS.
Add 2 mL of 0.1% Triton X-100 in PBS and incubate for 00:10:00 at Room temperature on a shaker to permeabilize the sample.
10m
Aspirate permeabilized solution and wash sample once with PBS.
Add 2 mL of blocking buffer (1% BSA + 0.1% Tween in 1X TBS) with a 00:10:00 incubation at Room temperature on a shaker.
10m
Aspirate blocking buffer and add a freshly prepared 2 mL primary antibody dilution (typically a 1:1000 dilution) made in blocking buffer.
Incubate the sample Overnight at 4 °C in the dark.
Aspirate the primary antibody solution and wash the sample once with 2 mL of blocking buffer.
Aspirate wash and add 2 mL of freshly prepared secondary antibody dilution (typically at a 1:2000 dilution) with a 00:10:00 incubation at Room temperature on a shaker in the dark.
10m
Aspirate the secondary antibody solution and wash the sample with 2 mL of blocking buffer with a 00:10:00 incubation at Room temperature on a shaker.
10m
Remove wash and cover sample with 2 mL PBS.
Proceed with fluorescence microscopy.
Samples may be stored at 4 °C protected from light for up to 72:00:00 ; bring dishes to Room temperature before imaging.
3d
Protocol references
● Begeman A, Smolka JA, Shami A, Waingankar TP, Lewis SC. A spatial atlas of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress. bioRxiv [Preprint]. 2024 Aug 17:2024.08.05.604215. doi: 10.1101/2024.08.05.604215. PMID: 39149346; PMCID: PMC11326164.
● Smolka JA, Lewis SC. In Situ Analysis of Mitochondrial DNA Synthesis Using Metabolic Labeling Coupled to Fluorescence Microscopy. Methods Mol Biol. 2023;2615:99-106. doi: 10.1007/978-1-0716-2922-2_8. PMID: 36807787.
Acknowledgements
We thank the Lewis Lab for helpful discussion.