Jul 18, 2024
Photo-Oxidation of MiniSOG Related to Cultured Neurons
- Daniela Boassa1,2,
- Thomas Steinkellner1,3,
- Matthew Madany1,2,
- Matthias G. Haberl1,2,4,
- Vivien Zell1,
- Carolina Li2,
- Junru Hu1,2,
- Mason Mackey1,2,
- Ranjan Ramachandra1,2,
- Stephen Adams1,2,5,
- Mark H. Ellisman1,2,
- Thomas S. Hnasko1,6
- 1Department of Neurosciences, University of California, San Diego, La Jolla, California;
- 2Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California;
- 3Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna;
- 4Charité−Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin;
- 5Department of Pharmacology, University of California, San Diego, La Jolla California;
- 6Research Service, VA San Diego Healthcare System, San Diego, California
- NCMIR@UCSD
External link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899175/
Protocol Citation: Daniela Boassa, Thomas Steinkellner, Matthew Madany, Matthias G. Haberl, Vivien Zell, Carolina Li, Junru Hu, Mason Mackey, Ranjan Ramachandra, Stephen Adams, Mark H. Ellisman, Thomas S. Hnasko 2024. Photo-Oxidation of MiniSOG Related to Cultured Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj63rplk5/v1
Manuscript citation:
Steinkellner T, Madany M, Haberl MG, Zell V, Li C, Hu J, Mackey M, Ramachandra R, Adams S, Ellisman MH, Hnasko TS, Boassa D. Genetic Probe for Visualizing Glutamatergic Synapses and Vesicles by 3D Electron Microscopy. ACS Chem Neurosci. 2021 Feb 17;12(4):626-639. doi: 10.1021/acschemneuro.0c00643. Epub 2021 Feb 1. PMID: 33522227; PMCID: PMC7899175.
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 25, 2022
Last Modified: July 18, 2024
Protocol Integer ID: 58787
Keywords: UCSD, NCMIR, Photo-oxidation, MiniSOG, miniSOG, glutamatergic synapse, glutamate, vesicular glutamate transporter (VGLUT2), neurotransmission, 3D electron microscopy (EM), deep learning, CDeep3M, genetic EM probe, ventral tegmental area (VTA), synaptic vesicles
Funders Acknowledgements:
NIH/National Institute of General Medical Sciences
Grant ID: R24GM137200
NIH/National Institute of Neurological Disorders and Stroke
Grant ID: U24NS120055
Abstract
This is a general protocol is for photo-oxidizing neuro-vesicle proteins with MiniSOG. We developed a genetically encoded probe to identify glutamatergic synaptic vesicles at the levels of both light and electron microscopy (EM) by fusing the mini singlet oxygen generator (miniSOG) probe to an intralumenal loop of the vesicular glutamate transporter-2.
Guidelines
We use the monomeric MiniSOG and not the td-miniSOG. The intrinsic fluorescence of MiniSOG is weak, and it photo-bleaches VERY quickly. In some cases, we have to rely on an additional fluorescent tag fused to MiniSOG to confirm expression. Therefore, we do not recommend exposures using regular lamps, but only with low power laser right before photo-oxidation. However, the photo-oxidation is not impaired after photo-bleaching.
Materials
List of buffers and solutions used:
- Fixative (2.5% Glut.,2.5 mM CaCl2 in 0.1 M Cacodyl. buffer pH7.4)
- Washing buffer: 0.1 M Cacodylate pH 7.4
- Blocking buffer: 50 mM Glycine, 10 mM KCN, 20mM aminotriazole in cacodylate buffer
- Fix 1% osmium tetra-oxide in 0.1 M Cacodyl. buffer
- DAB (free base - MW 214.27) (Cat. No. D8001-10G Sigma)
DAB solution (make 1 mL more than what is needed because some is lost during filtering):
- Dissolve 5.4 mg free base DAB in 1 mL 0.1 M HCl (final concentration 25.24 mM)
- Filter (0.22 um) into 9 mL of 0.1M Cacodylate or Blocking buffer
Before start
SET UP FOR PHOTO-OXIDATION
1.Filters we use for miniSOG
Leica Blue Excitation : 11513878
Ex:450-490
DM 510
Em:LP 515
Semrock Lucifer Yellow Filter : LuciferYellow-B-000 Filter Set
Ex:FF01-438/24
DM:F482-Di01
Em:FF01-538/40
Lamp House
We use 150W Xenon Lamp House. It is very strong and miniSOG can be oxidized with weaker lamp. It is a good idea to try photo-oxidation with your existing lamp house system. Just avoid UV excitation, which will cause DAB to precipitate.
490nm LED source may be good for miniSOG since it can excite miniSOG well. 1000mW 488 nm is very strong and should be enough or need to attenuate depend on how big illumination area is.
Objective Lens
Our microscope has three objectives:
63x Oil 1.30
63x Water 1.15
20x multi immersion 0.7
Plate cells in MaTek dishes.
Fix in 2.5% glutaraldehyde in 0.1 M cacodylate buffer at room temperature for 5 min. Then, for one hour on ice.
IMPORTANT: Keep Everything cold!
Wash 5x for 1 min in 0.1M cacodylate buffer.
Block for 30 min on ice with blocking buffer.
Wash 3x in 0.1M cacodylate buffer for 1 min.
Block in 5 – 10 mM mersalyl acid for 30 min.
Wash 3x for 1 min with 0.1M cacodylate buffer.
Remove solution from plate and replace with DAB solution.
Microscope
Microscope
At the microscope, set the temperature of the stage at 4˚C. Set the MaTek dish on the proper holder. Install oxygen, find target area with 63X objective lens, take initial picture (fluorescence + DIC).
Turn oxygen ON to blow onto surface of sample solution. (Regarding oxygenation: periodically replacing with freshly oxygenated solution is fine too when working with cultured cells).
Illuminate target area to photo-oxidize until light browning. Important: miniSOG is fast (2 to 10 minutes depending on protein). Optimal timing needs to be determined by investigator and will require the EM analysis of the samples at different reaction time.
Take final picture after photo-oxidation. Go to next area in the same dish and repeat, usually 3-4 areas per plate, making sure they are equally spaced to facilitate separation and cutting of individual blocks after polymerization.
When done, remove dish from holder, wash immediately 5x for 1 min in 0.1 M cacodylate buffer, on ice.
Fix in 1% osmium tetroxide in 0.1M cacodylate buffer. Leave 30 min on ice.
Wash 5x for 1 min in ddH2O.
Optional step: Add filtered 2% UA (uranyl acetate) in ddH2O at 4˚C for 1hr on ice. Wash 5x for 1 min in ddH2O.
Dehydrate: 20-50-70-90-100-100% cold Ethanol (1 min each). Then 100% dry ethanol at room temperature 3x for 1 minute.
Infiltration and Embedding: Prepare Durcupan: A:B:C:D=11.4:10:0.3:0.1 g
Add 50% ethanol: 50% Durcupan to dish. Leave for 30 min.
Pour out and add 100% Durcupan. Leave for 1 hour.
Add new 100% Durcupan resin, leave for 1 hr. Scrape out with a pair of plain wood applicators (Product No. 23-400-102 Fisherbrand) and without touching the cover slip containing the cells.
Add new 100% Durcupan resin, leave for 1 hr. Scrape out with a pair of plain wood applicators (Product No. 23-400-102 Fisherbrand) and without touching the cover slip containing the cells.
Add new 100% Durcupan resin, leave for 1 hr. Scrape out with a pair of plain wood applicators (Product No. 23-400-102 Fisherbrand) and without touching the cover slip containing the cells.
Add new 100% Durcupan resin, place in vacuum oven to polymerize for 48 hrs (60˚C).