TITLE: Fixation and imaging of HeLa cells after mitochondrial depolarization (Provisional unformatted)
AUTHORS: OLIVIA   HARDING, holzbaur  

[DESCRIPTION]
Ectopic expression of p62/SQSTM1 often induces puncta formation and poor cell health due to p62’s proclivity to multimerize and form filaments. We used the cell fixation protocol described here in order to over-express fluorescently tagged p62 at low levels in p62-/- cells and amplify the signal with immuno-labeling. By this method, we were able to express a variety of tagged p62 mutations to determine their effects on NEMO recruitment to depolarized mitochondria without introducing toxic artifacts of p62 overexpression. We also note the use of a modified fixation protocol to visualize various endogenous ATG8 proteins, which are also difficult to over-express in our system. Thus, fixation techniques are a critical complement to studies in live cells.

[STEPS]
1. 
2. Keywords
 Mitochondrial depolarization | paraformaldehyde fixation | confocal imaging
 Guidelines 
This protocol was adapted from a previous protocol for similar techniques
 (see dx.doi.org/10.17504/protocols.io.bujsnune
Here we use a primary antibody to a mitochondrial pr otein, HSP60, in
 order to identify mitochondria, instead of employing a genetically encoded
 fluorescent protein. While there are excellent mitochondrially targeted
 fluorescent proteins, using anti HSP60 allows us to demonstrate the
 versatility of the proto col to image both expressed, tagged proteins and
 immune labeled structures.
 Materials
3. 1.5 mL capped tubes (Millipore Sigma, EP022364120)
4. 10 mL conical tube (Corning CLS430055)
5. aluminum foil (Reynolds
6. Jewelers forceps, Dumont #5 (Millipore Sigma, F6521 1EA
7. Kimwipes Kimberly Clark Professional, 34155
8. Parafilm (Millipore Sigma, P7793 1EA
9. T ape
10. 15 cm cell culture dish (Millipore Sigma, CLS430599
11. Microscope sli d es (Fisher Scientific, 12 544 2)
12. VectaShield Plus (Vector Laboratories, H 1000 10
nail polish or similar sealant
 Reagents DMEMDMEM (Corning,(Corning, 1010–017017–CV)CV)

FBSFBS (HyClone)(HyClone)

GlutaMAXGlutaMAX glucoseglucose supplementsupplement (Gibco,(Gibco, 35050061)35050061)

DimethylDimethyl SulfoxideSulfoxide (DMSO)(DMSO) (Sigma,(Sigma, D650)D650)

EthanolEthanol

AntimycinAntimycin AA ((SigmaSigma–Aldrich,Aldrich, A8674)A8674) andand

OligomycinOligomycin AA ((SigmaSigma–Aldrich,Aldrich, 75351)75351)

PParaformaldehydearaformaldehyde (PFA)(PFA) (Millipore(Millipore Sigma,Sigma, 158127158127))

PhosphatePhosphate bufferedbuffered salinesaline (PBS)(PBS)

TritonTriton XX–100100 (Millipore(Millipore Sigma,Sigma, 11332481001)11332481001)

BovineBovine serumserum albuminalbumin (Fisher(Fisher BioReagents,BioReagents, BP1605BP1605–100)100)

antianti–HSP60,HSP60, pproducedroduced inin rabbitrabbit ((Sigma,Sigma, SAB4501464)SAB4501464)

antianti–p62/SQSTM1,p62/SQSTM1, producedproduced inin mousemouse (abcam,(abcam, ab56416ab56416))

GoatGoat antianti–RabbitRabbit IgGIgG (H+L)(H+L) HighlyHighly CrossCross–AdsorbedAdsorbed SecondarySecondary Antibody,Antibody, AlexaAlexa FluorFluor PlusPlus 405405 (ThermoFisher(ThermoFisher Scientific,Scientific, A48254)A48254)
 Goat
 Goat antianti–MouseMouse IgGIgG (H+L)(H+L) HighlyHighly CrossCross–AdsorbedAdsorbed SecondarySecondary Antibody,Antibody, AlexaAlexa Fluor™Fluor™ 546546 (ThermoFisher(ThermoFisher Scientific,Scientific, AA–1103011030
 Equipment

cellcell incubatorincubator toto maintainmaintain 3737 CC andand 5%5% CO2CO2 atmosphereatmosphere

vacuumvacuum apparatusapparatus

3737 CC waterwater bathbath

centrifugecentrifuge, refrigeratedrefrigerated

confocal microscope with confocal microscope with 60X objective 60X objective assocassociated softwareiated software
 Before start

TheThe startstart pointpoint forfor thisthis protocolprotocol isis afterafter cellscells growngrown onon glassglass coverslipcoverslipss inin aa 1212–wellwell plateplate havehave beenbeen transfectedtransfected withwith Parkin,Parkin, EGFPEGFP–NEMO,NEMO, HaloHalo–OPTN,OPTN, andand mCherrymCherry–p62p62 forfor 1818–2424 hourshours andand taggedtagged withwith HaloHalo ligandligand…

PreparePrepare 4545 mMmM sstocktock ofof AntimycinAntimycin AA byby suspendingsuspending 5050 mgmg solidsolid AntAAntA inin 22 mLmL ethanolethanol

PreparePrepare 1010 mMmM stockstock OligomycinOligomycin AA byby suspendingsuspending 55 mgmg solidsolid OligAOligA inin 630630 uLuL DMSODMSO

PreparePrepare 4%4% PFAPFA inin PBS.PBS. KeepKeep frozenfrozen atat --2020 CC
 o
 o Note:Note: PreparePrepare freshfresh forfor dayday–ofof fixationfixation oror thawthaw directlydirectly beforebefore ususee
 o
 o Note:Note: WillWill useuse 11 mLmL 4%4% PFAPFA perper wellwell

PreparePrepare CCultureulture MMediaedia byby makingmaking 10%10% FBS,FBS, 1%1% GlutaMAXGlutaMAX solutionsolution inin DMEMDMEM

PreparePrepare 0.5%0.5% TritonTriton XX–100100 inin PBSPBS (Permeabilization(Permeabilization buffer)buffer)
 o
 o Note:Note: PreparePrepare 0.750.75 mLmL PermeabilizationPermeabilization bufferbuffer perper well,well, storestore atat 44 CC
 o
 o Note:Note: BringBring toto roomroom temperaturetemperature beforebefore use.use. ThisThis willwill provideprovide lessless shockshock toto cells,cells, betterbetter preservingpreserving fixedfixed structuresstructures
 o
 o Note:Note: DoDo notnot useuse TritonTriton forfor permeabilizationpermeabilization ifif LC3LC3–autophagosomesautophagosomes areare thethe structurestructure ofof interest,interest, sincesince TritonTriton isis tootoo harsh.harsh. UseUse iceice coldcold methanolmethanol ifif imagingimaging LC3LC3 structures.structures.

PreparePrepare 0.2%0.2% TritonTriton XX–100100/3%/3% BSABSA inin PBSPBS (Blocking(Blocking buffer)buffer)
 o
 o Note:Note: PreparePrepare 0.750.75 mLmL BlockingBlocking bufferbuffer perper well,well, storestore atat 44 CC
 o
 o Note:Note: BringBring toto roomroom temperaturetemperature beforebefore useuse

PreparePrepare aa humidityhumidity chamberchamber asas previouslypreviously describeddescribed ((dx.doi.org/10.17504/protocols.io.bujsnunedx.doi.org/10.17504/protocols.io.bujsnune))
 Step-by-step
 AntA/OligA treatment

PreparePrepare workingworking AntA/OligAAntA/OligA solutionsolution byby trantransferringsferring .5.5 mLmL conditionedconditioned mediamedia toto aa 1.51.5 mLmL tubetube andand addingadding .25.25 uLuL 1010 mMmM OligAOligA andand 22 mLmL 4545 mMmM AntAAntA

GentlyGently dropdrop workingworking AntA/OligAAntA/OligA solutionsolution ontoonto cellscells

IncubateIncubate atat 3737 C,C, 5%5% CO2CO2 forfor 5555–6565 minmin
 o
 o Note:Note: ourour protocolprotocol callscalls forfor ~~11 hrhr mitochondrialmitochondrial damagedamage sincesince thatthat waswas ourour timecoursetimecourse ofof interest.interest. UsersUsers maymay varyvary thethe timecoursetimecourse inin orderorder toto examineexamine earlierearlier oror laterlater effectseffects ofof globalglobal mitochondrialmitochondrial damage.damage.
 o
 o Note:Note: toto carrycarry outout vehiclevehicle controlcontrol experiment,experiment, useuse thethe equivalentequivalent amountsamounts ofof ethanoethanoll andand DMSODMSO treatmenttreatment
 Fixation

2525 minutesminutes beforebefore AntA/OligAAntA/OligA treatmenttreatment isis completecomplete, warmwarm 4%4% PFAPFA andand 1X1X PBSPBS toto 3737 CC

RemoveRemove cellscells fromfrom incubatorincubator andand aspirateaspirate mediamedia
 o
 o Note:Note: whenwhen possible,possible, keepkeep cellscells coveredcovered withwith aa sheetsheet ofof aluminumaluminum foil,foil, sincesince fluorescentfluorescent liganligandsds areare lightlight–sensitivesensitive

QuicklyQuickly dropdrop onon 0.50.5 mLmL warmedwarmed 1X1X PBSPBS

AspirateAspirate PBSPBS

RepeatRepeat warmwarm PBSPBS washwash

DropDrop onon 11 mLmL warmedwarmed 4%4% PFAPFA

IncubateIncubate atat 3737 CC forfor 1010 minmin
 o
 o Note:Note: warmedwarmed PBSPBS andand 4%4% PFAPFA needneed notnot bebe sterile.sterile. Thus,Thus, ifif cellscells areare nono longerlonger sterile,sterile, incubateincubate inin aa nonnon–sterilesterile 3737 CC environmentenvironment

DropDrop onon 0.50.5 mLmL warmedwarmed PBSPBS

AspirateAspirate PBSPBS

DropDrop onon 0.50.5 mLmL warmedwarmed PBSPBS

IncubateIncubate coveredcovered atat roomroom temperaturetemperature forfor 33 minmin
 o
 o Note:Note: cellscells cancan bebe coveredcovered andand storedstored atat 44 CC forfor upup toto severalseveral daysdays afterafter thisthis step.step. IfIf youyou choosechoose toto pausepause atat thisthis step,step, addadd atat leastleast 1.51.5 mLmL PBSPBS toto wellswells toto preventprevent evaporationevaporation
 Permeabilization

AspirateAspirate PBSPBS

DropDrop onon 0.750.75 mLmL PermeabilizationPermeabilization bufferbuffer

IncubateIncubate coveredcovered atat roomroom temperaturetemperature forfor 55 minmin
 o
 o Note:Note: IfIf fixationfixation protocolprotocol isis usedused toto examineexamine autophagosomalautophagosomal structures,structures, ddoo notnot useuse bufferbuffer withwith TritonTriton, asas thisthis willwill destroydestroy thethe vesicles.vesicles. Instead,Instead, useuse iceice coldcold methanolmethanol permeabilizationpermeabilization bufferbuffer andand goatgoat serum/BSAserum/BSA blockingblocking buffer.buffer.
 Blocking

AddAdd 250250 uLuL BlockingBlocking bufferbuffer toto eacheach wellwell

IncubateIncubate coveredcovered atat roomroom temperaturetemperature forfor 4545 minmin
 o
 o NNote:ote: BlockingBlocking stepstep cancan bebe upup toto 11 hrhr
 o
 o Note:Note: prepareprepare primaryprimary antibodyantibody dilutiondilution duringduring thisthis stepstep

AspirateAspirate BlockingBlocking bufferbuffer
 Primary antibody

prepareprepare 200200 uLuL PrimaryPrimary antibodyantibody dilutiondilution perper coverslip,coverslip, withwith 1:1:250250 antianti–HSP60HSP60 andand 1:5001:500 antianti–p62p62 inin BlockingBlocking bufferbuffer
 o
 o Note:Note: beforebefore use,use, spinspin primaryprimary antibodyantibody atat toptop speedspeed inin aa refrigeratedrefrigerated centrifugecentrifuge forfor 55 min.min. PipetPipet fromfrom toptop ofof solutionsolution toto ensureensure thatthat nono aggregatesaggregates areare presentpresent
 o
 o Note:Note: whilewhile mCherrymCherry–p62p62 waswas transfectedtransfected inin thesethese cells,cells, antianti–p62p62 antibodyantibody bboostsoosts thethe signalsignal

UseUse sharpsharp forcepsforceps toto carefullycarefully liftlift thethe coverslipcoverslip outout ofof thethe wellwell andand dabdab excessexcess BlockingBlocking bufferbuffer onon aa KimwipeKimwipe

placeplace slipslip cellcell–sideside upup onon ParafilmParafilm inin thethe humidityhumidity chamberchamber

fromfrom thethe edgeedge ofof thethe coverslip,coverslip, pipetpipet primaryprimary antibodyantibody dilutiondilution ontoonto thethe cellscells

incubateincubate coveredcovered atat 44 CC overnightovernight
 o
 o Note:Note: twisttwist twotwo KimwipesKimwipes andand wetwet withwith waterwater soso thatthat theythey areare moremore thanthan dampdamp butbut notnot dripping.dripping. PackPack thethe wetwet KimwipesKimwipes intointo thethe edgesedges ofof thethe humidityhumidity chamberchamber inin orderorder toto preventprevent evaporationevaporation ofof antibodyantibody solutionsolution duduringring thethe incubationincubation

1818–2424 hourshours later,later, aaspiratespirate antibodyantibody dilutiondilution fromfrom thethe edgeedge ofof thethe coverslip,coverslip, gentlygently liftinglifting oneone sideside ofof thethe slipslip withwith forcepsforceps ifif necessarynecessary toto allowallow bufferbuffer toto slideslide offoff

fromfrom thethe edgeedge ofof thethe coverslip,coverslip, pipetpipet 100100 uLuL roomroom temperaturetemperature PBSPBS ontoonto thethe cellscells

incubateincubate coveredcovered atat roomroom temperaturetemperature forfor 55 minmin

AspirateAspirate PBSPBS fromfrom thethe edgeedge ofof thethe coverslip,coverslip, gentlygently liftinglifting oneone sideside ofof thethe slipslip withwith forcepsforceps ifif necessarynecessary toto allowallow bufferbuffer toto slideslide offoff

repeatrepeat previousprevious threethree stepssteps forfor aa totaltotal ofof fourfour washeswashes
 Secondary antibody

PreparePrepare secondarysecondary antibodyantibody solutionsolution byby dilutingdiluting goatgoat antianti–rabbitrabbit 404055 andand goatgoat antianti–mousemouse 1:2001:200 inin BlockingBlocking bufferbuffer
 o
 o Note:Note: prepareprepare 125125 uLuL secondarysecondary solutionsolution forfor eacheach coverslipcoverslip
 o
 o Note:Note: beforebefore use,use, spinspin primaryprimary antibodyantibody atat toptop speedspeed inin aa refrigerrefrigeratedated centrifugecentrifuge forfor 55 min.min. PipetPipet fromfrom toptop ofof solutionsolution toto ensureensure thatthat nono aggregatesaggregates areare presentpresent

fromfrom thethe edgeedge ofof thethe coverslip,coverslip, pipetpipet secondarysecondary antibodyantibody dilutiondilution ontoonto thethe cellscells

IncubateIncubate coveredcovered atat roomroom temperaturetemperature forfor 4545 minmin
 o
 o Note:Note: secondarysecondary incubationincubation cancan bebe upup toto 11 hrhr
 o
 o Note:Note: warmwarm VectaShieldVectaShield toto roomroom temperaturetemperature duringduring thisthis stepstep

AspirateAspirate antibodyantibody dilutiondilution fromfrom thethe edgeedge ofof thethe coverslip,coverslip, gentlygently liftinglifting oneone sideside ofof thethe slipslip withwith forcepsforceps ifif necessarynecessary toto allowallow bufferbuffer toto slideslide offoff

fromfrom thethe edgeedge ofof thethe coverslip,coverslip, pipetpipet 100100 uLuL roomroom temperaturetemperature PBSPBS ontoonto thethe cellscells

incubateincubate coveredcovered atat roomroom temperaturetemperature forfor 55 minmin

AspirateAspirate PBSPBS fromfrom thethe edgeedge ofof thethe coverslip,coverslip, gentlygently liftinglifting oneone sideside ofof thethe slipslip withwith forcepsforceps ifif necessarynecessary toto allowallow bufferbuffer toto slideslide offoff

repeatrepeat previousprevious threethree stepssteps forfor aa totaltotal ofof fourfour washeswashes
 Mounting

pipetpipet 12.512.5 uLuL roomroom temperaturetemperature VeVectaShieldctaShield ontoonto aa microscopemicroscope slideslide

PickPick upup coverslipcoverslip withwith forcepsforceps andand dabdab excessexcess PBSPBS ontoonto aa KimwipeKimwipe

LayLay coverslipcoverslip cellcell–sideside downdown ontoonto VectaShieldVectaShield dropdrop

SealSeal edgesedges ofof coverslipcoverslip withwith nailnail polishpolish

LayLay flatflat untiluntil setset
 o
 o Note:Note: storestore slidesslides atat 44 CC
 Imaging

UsingUsing 60X60X objectiveobjective andand RFPRFP epifluorescence,epifluorescence, findfind thethe focalfocal planeplane ofof fixedfixed cellscells byby lookinglooking forfor dsRed2dsRed2–labeledlabeled mitochondriamitochondria
 o
 o Note:Note: WeWe findfind thatthat 60X60X isis sufficientsufficient magnificationmagnification toto collectcollect severalseveral cellscells inin eacheach fieldfield ofof viewview withwith enoughenough resolutionresolution toto performperform analysisanalysis aandnd quantification.quantification. OtherOther magnificationsmagnifications maymay bebe appropriateappropriate forfor variousvarious applications.applications.

ConfigureConfigure 405,405, 488,488, 561,561, andand 647647 laserslasers andand accompanyingaccompanying exposureexposure timestimes toto maximizemaximize dynamicdynamic rangerange ofof eacheach signalsignal
 o
 o Note:Note: sincesince thethe cellscells areare fixed,fixed, therethere isis lessless downsidedownside ttoo photobleachingphotobleaching thethe samples.samples.

ConfigureConfigure collectioncollection parametersparameters toto recordrecord aa >2>2 umum ZZ stackstack ofof images.images.
 o
 o Note:Note: onon ourour system,system, 0.150.15 umum isis thethe minimumminimum stepstep size,size, thusthus 2121 sectionssections isis sufficient.sufficient.

CollectCollect imagesimages fromfrom fieldfieldss ofof viewview withwith multiplemultiple cellscells exhibitingexhibiting allall visiblevisible tags.tags. CollectCollect volumevolume fromfrom thethe 5050thth toto 7575thth percentilepercentile ofof thethe cellcell (see(see FigureFigure 1).1). IfIf mitochondriamitochondria areare depolarized,depolarized, cancan useuse OPTNOPTN signalsignal (recruitment(recruitment toto fragmentedfragmented mitochondria)mitochondria) toto determinedetermine whetherwhether ParkinParkin isis expressedexpressed…
 Figure 1. Schematic of HeLa cell volume. Dotted lines indicate target volume to image.

CollectCollect imagesimages inin orderorder toto recordrecord 1010–2020 cellscells perper condition,condition, dependingdepending onon thethe qualityquality ofof thethe transfectiontransfection andand cells.cells. CollectCollect imagesimages fromfrom diversediverse areasareas ofof thethe coverslip.coverslip.