TITLE: Fixing hippo neurons to assess endogenous NEMO during oxidative stress    
AUTHORS: OLIVIA HARDING

[DESCRIPTION]
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.  

[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. 

[STEPS]
SECTION: Plating and maintenance of hippocampal neurons
1. 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. 
 

SECTION: Plating and maintenance of hippocampal neurons
4. Add AraC (5 uM) one day post-plating to prevent glial proliferation
SECTION: Plating and maintenance of hippocampal neurons
3. 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. 
SECTION: Transfection of primary neurons
6. 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. 
SECTION: Mitophagy induction in neurons
10. 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. 
SECTION: Fluorescent labeling in neurons
8. Incubate neurons in 2 uM SNAP ligand (Janelia-Fluor 646) for 30 min. 
SECTION: Fix, permeabilize, and block neurons
12. Gently add at least 0.5 mL fixative to each dish and incubate 10 min at 37 degrees
SECTION: Plating and maintenance of hippocampal neurons
2. Incubate neurons for at least 2 but no more than 5 hours at 37 degrees C, 5% CO2. 
SECTION: Fluorescent labeling in neurons
9. Wash 2X, then rest 30 min in conditioned media. 
SECTION: Fix, permeabilize, and block neurons
11. Prepare 4% paraformaldehyde (PFA)/4% sucrose in PBS 
SECTION: Imaging
27. 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). 
SECTION: Mitophagy induction in neurons
10.1. During this period, prepare fixation reagent (see next step)
SECTION: Fix, permeabilize, and block neurons
11.1. Thaw a 10 mL aliquot of 4% PFA in PBS using a 37 degree water bath
SECTION: Fix, permeabilize, and block neurons
11.2. Add 0.4 g sucrose to aliquot
SECTION: Fix, permeabilize, and block neurons
11.3. Rotate at R.T. for 10 min to dissolve
SECTION: Fix, permeabilize, and block neurons
11.4. Warm 4% PFA/4% sucrose to 37 degrees
SECTION: Fix, permeabilize, and block neurons
13. Aspirate PFA and wash samples with PBS 4X 5 min
SECTION: Fix, permeabilize, and block neurons
14. Permeabilize cells by adding at least 300 uL 0.2% Triton-X in PBS for 5 min 
SECTION: Perform immunolabeling and Hoechst stain
16. 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. 
SECTION: Fix, permeabilize, and block neurons
15. Block cells with at least 300 uL BSA block (see Materials) for 50 min
SECTION: Fix, permeabilize, and block neurons
15.1. Prepare primary antibody solution during this step.
SECTION: Perform immunolabeling and Hoechst stain
17. Aspirate blocking solution from each sample and gently add 180 uL primary solution. 
SECTION: Perform immunolabeling and Hoechst stain
18. Incubate samples in primary solution overnight in a dark environment.
SECTION: Perform immunolabeling and Hoechst stain
19. 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. 
SECTION: Perform immunolabeling and Hoechst stain
20. 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.
SECTION: Perform immunolabeling and Hoechst stain
16.1. Centrifuge primary antibody at 4 degrees, max speed, for 4 min before use. After centrifugation, pipet from top of antibody aliquot.  
SECTION: Perform immunolabeling and Hoechst stain
20.1. Centrifuge primary antibody at 4 degrees at max speed for 4 min before use. After centrifugation, pipet from top of antibody aliquot.  
SECTION: Perform immunolabeling and Hoechst stain
21. 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. 
SECTION: Perform immunolabeling and Hoechst stain
22. Aspirate secondary solution and add 100-200 uL PBS to wash for 5 min. Carry out PBS wash for a total of 2 times. 
SECTION: Perform immunolabeling and Hoechst stain
23. Prepare Hoechst solution with a volume of 110 uL * number of dishes. Use Hoechst dye at 1:1000 dilution in PBS.
SECTION: Perform immunolabeling and Hoechst stain
24. Aspirate PBS from second wash and add Hoechst solution. Incubate 5 min at room temperature. 
SECTION: Perform immunolabeling and Hoechst stain
25. Repeat step 22. 
SECTION: Perform immunolabeling and Hoechst stain
26. Aspirate final PBS wash and gently add enough volume of Vectashield to cover surface of glass window. 
SECTION: Transfection of primary neurons
7. Incubate transfected neurons 36-48 hours. 
SECTION: Transfection of primary neurons
6.1. Equilibrate a volume of fresh MM equal to 2 mL per dish by incubating MM in a flask in the cell culture incubator.
SECTION: Transfection of primary neurons
6.2. Replace conditioned MM with 1 mL fresh MM for each dish. Save conditioned MM in a fresh flask in the incubator.
SECTION: Transfection of primary neurons
6.3. 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.
SECTION: Transfection of primary neurons
6.4. 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. 
SECTION: Transfection of primary neurons
6.5. Add A+B (300 uL) to each dish of primary neurons dropwise. 
SECTION: Transfection of primary neurons
6.6. Incubate at 37 degrees 45 min.
SECTION: Transfection of primary neurons
6.7. Replace transfection media with 50%/50% fresh and conditioned media.
SECTION: Plating and maintenance of hippocampal neurons
5. Allow neurons to grow until Day 6.