Jul 31, 2023
Fixing hippo neurons to assess endogenous NEMO during oxidative stress
Forked from Investigation_of_mitophagy_in_Hippo_neurons
- 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: OLIVIA HARDING 2023. Fixing hippo neurons to assess endogenous NEMO during oxidative stress . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjx8dlmk/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: May 30, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82638
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: Mechanisms of mitochondrial damage control by PINK1 and Parkin (ASAP-000350)
Abstract
We altered a previously developed method (dx.doi.org/10.17504/protocols.io.bxpfpmjn) in order to investigate the interaction of endogenous NEMO with damaged mitochondria in Hippocampal rat neurons. Mitochondrial damage was induced by the administration of 35 nM Antimycin A, a complex III inhibitor, over 1 hr. Here, we visualized neuronal mitochondria with a mito-targeted construct, Mito-SNAP, and a fluorescent SNAP ligand, JaneliaFluor 646, though other mitochondrial markers may be suitable. Endogenous NEMO was visualized with the commercially available anti-NEMO primary antibody (abcam). AntA-treated neurons exhibited fragmented mitochondria. We did not observe a change in the appearance of NEMO labeling in AntA treated cells versus non-treated cells.
Materials
BSA block: 0.2% Triton-X/3% BSA in PBS.
Before start
Prepare poly-L-lysine (PLL)-coated surfaces on which to plate hippocampal neurons by pipetting 700-1000 uL 0.5 mg/mL PLL onto glass-bottomed dishes, prioritizing coverage of the glass imaging window. Neurons may also be fixed on glass coverslips.
Plating and maintenance of hippocampal neurons
Plating and maintenance of hippocampal neurons
6d
6d
Plate primary hippocampal Sprague Dawley rat neurons (embryonic day 18) on 35 mm glass-bottomed, PLL-coated dishes at 250,00 cells per dish in MEM supplemented with 10% horse serum, 33 mM D-glucose, and 1 mM sodium pyruvate. This is day 0.
Note
Information for dissection and separation of hippocampal neurons is not included because we outsource these steps to an on-campus core facility.
Incubate neurons for at least 2 but no more than 5 hours at 37 degrees C, 5% CO2.
2h
Replace media with Neurobasal (Gibco) supplemented with 33 mM D-glucose, 2 mM GlutaMAX (Invitrogen), 100 units/ml penicillin, 100 ug/ml streptomycin, and 2% B-27 (ThermoFisher) (Maintenance Media; MM) and continue to maintain at 37 degrees C, 5% CO2.
1d
Add AraC (5 uM) one day post-plating to prevent glial proliferation
Allow neurons to grow until Day 6.
Transfection of primary neurons
Transfection of primary neurons
2d
2d
On day 6, transfect neurons. 0.3 ug Mito-SNAP per 35-mm dish. Use 3.8 uL Lipofectamine 2000 Transfection Reagent (ThermoFisher) per dish.
2h
Equilibrate a volume of fresh MM equal to 2 mL per dish by incubating MM in a flask in the cell culture incubator.
45m
Replace conditioned MM with 1 mL fresh MM for each dish. Save conditioned MM in a fresh flask in the incubator.
Prepare transfection solutions A and B by adding 0.3 ug Mito-SNAP to 150 uL Neurobasal (A) and 3.8 uL Lipofectamine 2000 to 150 uL Neurobasal (B) for each dish. Rotate by hand 8 times and incubate at room temperature for 5 min.
5m
Briefly centrifuge A and B with a tabletop centrifuge. Then combine A and B and rotate by hand 8 times to mix. Incubate at room temperature for 20 min.
20m
Add A+B (300 uL) to each dish of primary neurons dropwise.
Incubate at 37 degrees 45 min.
45m
Replace transfection media with 50%/50% fresh and conditioned media.
Incubate transfected neurons 36-48 hours.
2d
Fluorescent labeling in neurons
Fluorescent labeling in neurons
1h
1h
Incubate neurons in 2 uM SNAP ligand (Janelia-Fluor 646) for 30 min.
30m
Wash 2X, then rest 30 min in conditioned media.
30m
Mitophagy induction in neurons
Mitophagy induction in neurons
1h
1h
Replace neuron media with MM containing 35 nM Antimycin A (or, for control conditions, media containing no small molecule inhibitor) and incubate for 1 hr.
1h
During this period, prepare fixation reagent (see next step)
Fix, permeabilize, and block neurons
Fix, permeabilize, and block neurons
35m
35m
Prepare 4% paraformaldehyde (PFA)/4% sucrose in PBS
Thaw a 10 mL aliquot of 4% PFA in PBS using a 37 degree water bath
7m
Add 0.4 g sucrose to aliquot
Rotate at R.T. for 10 min to dissolve
10m
Warm 4% PFA/4% sucrose to 37 degrees
10m
Gently add at least 0.5 mL fixative to each dish and incubate 10 min at 37 degrees
10m
Aspirate PFA and wash samples with PBS 4X 5 min
20m
Permeabilize cells by adding at least 300 uL 0.2% Triton-X in PBS for 5 min
5m
Block cells with at least 300 uL BSA block (see Materials) for 50 min
Prepare primary antibody solution during this step.
Perform immunolabeling and Hoechst stain
Perform immunolabeling and Hoechst stain
2d
2d
Prepare primary solution with a volume of 200 uL * number of dishes. Use anti-NEMO (abcam, raised in rabbit) at a 1:500 dilution in BSA block.
Centrifuge primary antibody at 4 degrees, max speed, for 4 min before use. After centrifugation, pipet from top of antibody aliquot.
Aspirate blocking solution from each sample and gently add 180 uL primary solution.
Incubate samples in primary solution overnight in a dark environment.
1d
The following day, aspirate primary solution and add 100-200 uL PBS to wash for 5 min. Carry out PBS wash for a total of 4 times.
20m
Prepare secondary solution with a volume of 110 uL * number of dishes. Use anti-rabbit 488 secondary antibody at 1:200 dilution in BSA block.
Centrifuge primary antibody at 4 degrees at max speed for 4 min before use. After centrifugation, pipet from top of antibody aliquot.
Aspirate PBS from final wash and add 100 uL secondary solution to each sample, making sure to submerge all areas of the glass window. Incubate 1 hr at room temperature.
1h
Aspirate secondary solution and add 100-200 uL PBS to wash for 5 min. Carry out PBS wash for a total of 2 times.
10m
Prepare Hoechst solution with a volume of 110 uL * number of dishes. Use Hoechst dye at 1:1000 dilution in PBS.
Aspirate PBS from second wash and add Hoechst solution. Incubate 5 min at room temperature.
5m
Repeat step 22.
10m
Aspirate final PBS wash and gently add enough volume of Vectashield to cover surface of glass window.
Imaging
Imaging
Image samples, for example, with a Nikon Eclipse Ti Microscope with a 100X objective (Apochromat, 1.49-N.A. oil immersion) and an UltraView Vox spinning disk confocal system (PerkinElmer). Collect Z-stacks at 0.15 nm/step with Volocity acquisition software (PerkinElmer).