Dec 09, 2025
Sample Preparation for FLASH-PAINT
- Devin M. Fuller1,2,
- Florian Scheuder1,
- Thomas Melia1,2
- 1Department of Cell Biology, Yale University School of Medicine, New Haven, CT;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, 20 MD
Protocol Citation: Devin M. Fuller, Florian Scheuder, Thomas Melia 2025. Sample Preparation for FLASH-PAINT. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11ry7vr2/v1
Manuscript citation:
Fuller Devin M, Wu Yumei, Schueder Florian, Rasool Burha, Nag Shanta, Korfhage Justin L, Garcia-Milian Rolando, Melnyk Katerina D, Bewersdorf Joerg, De Camilli Pietro, Melia Thomas J (2025) ATG2A engages RAB1A and ARFGAP1 positive membranes during autophagosome biogenesis eLife 14:RP107316
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: December 01, 2025
Last Modified: December 09, 2025
Protocol Integer ID: 233893
Keywords: using flash, paint, sample, flash, protocol
Funders Acknowledgements:
National Institutes of Health
Grant ID: R01 GM100930
National Institutes of Health
Grant ID: R35 GM153482
National Institutes of Health
Grant ID: F31 AG079606
Aligning Science Across Parkinson’s
Grant ID: ASAP-025173
Abstract
A protocol in how to prepare and image your samples using FLASH-PAINT
Materials
The
following reagents were purchased for FLASH-PAINT: Cy3B-modified DNA
oligonucleotides were custom-ordered from IDT. Sodium chloride 5 M (cat:
AM9759) were obtained from Ambion. Ultrapure water (cat: 10977-015) was
purchased from Invitrogen. µ-Slide 8-well chambers (cat: 80807) were purchased from ibidi. Methanol
(cat: 9070-05) was purchased from J.T. Baker. Glycerol (cat: G5516-500ml),
protocatechuate 3,4-dioxygenase pseudomonas (PCD) (cat: P8279),
3,4-dihydroxybenzoic acid (PCA) (cat: 37580-25G-F) and (+−)-6-hydroxy-2,5,7,8-
tetra-methylchromane-2-carboxylic acid (Trolox) (cat: 238813-5 G) were ordered
from Sigma. 1× Phosphate Buffered Saline (PBS) pH 7.2 (cat: 10010-023) was
purchased from Gibco. Paraformaldehyde (cat: 15710) were obtained from Electron
Microscopy Sciences. Bovine serum albumin (cat: 001-000-162) was ordered from
Jackson ImmunoResearch. Triton X-100 (cat: T8787-50ML) was purchased from
Sigma-Aldrich. DNA labeled Nanobodies were obtained from Massive Photonics.
Troubleshooting
Sample Preparation Protocol
Fix cells with fresh 3% PFA for 30 min at RT.
Immobilization and block with 3% BSA and 0.1% Triton-X.
Incubate sample with the desired antibodies with the lowest binding efficiency, one per organism in blocking solution (3% BSA + 0.1% Triton-X) overnight at 4°C (1:300 dilution as always). For example, we incubated our samples with the GFP Nanobody, ATG13 AB (rabbit) and WIPI2 AB (mouse).
For the remainder of the antibodies, prepare a preincubation: Mix 0.5 µL of primary AB with 5µL of 1xPBS and 2µL of nanobody in a PCR tube and let it incubate overnight at 4°C. The following substeps illustrate the antibodies that we used with their corresponding nanobody (NB), each of which was conjugated to a unique DNA barcode:
0.5 µL of LC3B-AB + 2 µL of 2nd NB-A20 + 5 µL 1xPBS
0.5 µL of ATG9A-AB + 2 µL of 2nd NB-A5 + 5 µL 1xPBS
0.5 µL of P62-AB + 2 µL of 2nd NB-A36 + 5 µL 1xPBS
0.5 µL of NBR1-AB + 2 µL of 2nd NB-A19 + 5 µL 1xPBS
0.5 µL of FIP200-AB + 2 µL of 2nd NB-A8 + 5 µL 1xPBS
0.5 µL of ATG13-AB + 2 µL of 2nd NB-A15 + 5 µL 1xPBS
0.5 µL of Syntaxin17 + 2 µL of 2nd NB-A38 + 5 µL 1xPBS
0.5 µL of ARFGAP1-AB + 2 µL of 2nd NB-A10 + 5 µL 1xPBS
0.5 µL of LAMP1-AB + 2 µL of 2nd NB-A39 + 5 µL 1xPBS
On the next day block incubate sample with the 2nd nanobodies corresponding to the antibodies that we incubated in the sample overnight for 1 hour at room temperature (e.g. NB-A25 26 2nd NB-A27 corresponding to ATG13 AB and WIPI2 AB).
Also on the next day block PCR tube mixes with 4 µL of unlabeled NB for 5 min.
Pool the content of all PCR tubes and put it on the sample for 2.5 h at room temperature.
Wash four times with 1x PBS for 30 seconds, 1 minute, and twice for 5 minutes each.
Post-fix the cells with 3% PFA and 0.1% GA for 10 minutes.
Wash four times with 1x PBS for 30 seconds, 1 minute, and twice for 5 minutes each.
Imaging on the microscope
Introducing the desired adapter (for example, A19-R2 (20 nM) to image NBR1) and the imager R2 (1 nM) in Buffer C.
After image acquisition, rinse 3 times with 1× PBS.
Erase: Add 200 µL of E19-R2 eraser (100 nM in Buffer C) to the sample and incubate for 5 min.
Next, introduce the adapter for the second target, e.g. FIP200: use A8-R2 (20 nM) and the R2 imager (1 nM).
After image acquisition, rinse 3 times with 1× PBS.
Erase: Add 200 µL of E8-R2 eraser (100 nM in Buffer C) to the sample and incubate for 5 min.
Iterate this process until all targets are imaged.
Protocol references
Acknowledgements
This work was supported by grants from the National Institutes of Health (R01 GM100930 and R35 GM153482 to TJM; R01 GM151829 to JB; DA018343 to PDC), F31 AG079606 to DMF and F31 DK136246 to JLK. This research was also funded in part through Aligning Science Across Parkinson’s (ASAP-025173 to TJM and PDC) through the Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Howard Hughes Medical Institute (HHMI; PDC). FS acknowledges support from the Human Frontier Science Program (LT000056/2020-C). JB acknowledges support by the Wellcome Leap Foundation. Imaging was supported by the Yale Center for Cellular and Molecular Imaging (both the fluorescence and electron microscopy facilities). We also thank the MS & Proteomics Resource at Yale University for providing the necessary mass spectrometers and the accompany biotechnology tools funded in part by the Yale School of Medicine and by the Office of The Director, National Institutes of Health (S10OD02365101A1, S10OD019967, and S10OD018034). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.