Pla$ormforskeletalmuscle0ssueclearingforfluorescentreportersandimmunofluorescencestaining.

BhavaniSaiRohitMurakondaa,b,c,,MayankVermaa,b,c,YokoAsakuraa,b,c,AtsushiAsakuraa,b,cStemCellIns>tutea,Paul&SheilaWellstoneMuscularDystrophyCenterb,DepartmentofNeurologyc,UniversityofMinnesotaMedicalSchool.Minneapolis,MN,USA,55455

Introduc0on

CurrentResearch

Workflow

Conclusions

FutureExperiments

Acknowledgements

HighThroughputReagentScreeningofputa0veclearingreagents

Preserva0onofFluorescence ThinSamplePrep

Skeletalmuscle is a highly ordered yet complex>ssue containing several celltypes that interact with each other in order to maintain structure andhomeostasis.Itiscomposedofseveraldifferenttypesofcellsincludingmusclefibers, satellite cells, blood vessels, fibroblasts, immune cells, etc. It is also ahighly regenera>ve >ssue that responds to damage in a highly intricate butstereotypicmanner,withdis>nctspa>alandtemporalkine>cs.Therearemanycellular interac>ons that occur within the >ssue, such as the interac>onbetween satellite cells and blood vessel endothelial cells. Typically, cross-sec>onsor longitudinal sec>ons, obtained from frozenor paraffinembedded>ssue blocks, are immunolabled and a single slice is examined under themicroscope.Whilethisisinforma>veinthelateralplane,theinforma>onintheaxial direc>on is lost leading to underrepresenta>on ormisrepresenta>on ofthe actual data. As such, it is important to obtain informa>on from all threedimensionsinthe>ssuetostudytheanatomicalandcellularinterac>onsinthe>ssue.However,theseinterac>onsaremosteffec>velystudiedbyimagingthe>ssue in its na>ve conforma>on. One approach to studying the spa>alinterac>on of cells in their three dimensional (3D) anatomical state, is byobtaining serial sec>ons, imaging each sec>on and reconstruc>ng the 3Drepresenta>on through manual or automated methods. But, this process istechnicallychallenging,errorproneandlaborintensive.Forthisreason,wholemountimagingisthemosteffec>vewaytoobservetheinterac>ons.However,lightscaYeringduetolipidsandproteinsinthe>ssuehindereffec>veimagingthroughthewhole>ssue.

Recentprotocolsfor3Dimagingofwholebrain>ssue(Costan>ni,Irene,etal.2015,Scien&ficreports).However,theseprotocolsfocusonclearingneuronalbrain>ssuewhichhasadras>callydifferentcomposi>oncomparedtoskeletalmuscle.Theaimofthisprojectistocomeupwitha>ssueclearingprotocoldesignedforproteinrichskeletalmusclewhichmaydifferfromlipidrichneuronal>ssue.

•  Homogenizedmuscleisusedinordertochecktheclearingefficiencyofthereagentsinahighthroughputmanner

Highthroughputreagentscreening

•  Checkthecompa>bilityofthereagentswithfluorescencereporters

Compa>bilitywithfluorescencereporter

•  Screenthereagents’effectonan>bodyspecificity/sensi>vityincryosec>ons.Thismaybeimportanttotestforeach

an>bodyImmunolabeling

•  Observetheclearingefficencyandpreserva>onoffluorescenceinthethin>ssuesamplesusingdeconvolu>on

microscopyThinsampleclearing

•  Theul>mategoaloftheprojectistoimagewholemuscle>ssuewithfluorescentreportersandimmunolabeling.LargeSamples

Cryosec>ons of muscle with fluorescent reporters were incubated with theputa>veclearingreagents.Ifthereagentpreservedfluorescentreporters,thenitcanbeusedtoobservemanyinterac>ons,throughfluorescentimaging.Forexample,methanoldoesn’tpreservefluorescenceandthereforewouldnotaidinfluorescentanalysisviastaining.

Inordertotesttheclearingefficiencyinmusclesamples,Thincremastermusclewereclearedandassessedbydeconvolu>onmicroscopy.Theclearingefficiencyofthereagentsandthepreserva>onoffluorescencewasassessedbyevalua>ngthesignaltonoisera>o(SNR)usingImageJ.

•  Screenaddi>onalcompounds•  Highthroughputscreeningoffluorescencepreserva>on•  Combinecompoundstoproduceacocktailcapableofeffec>velyclearing

muscle>ssueforimaging•  Testthecompoundsonlargermusclesamples.

Cryosec>onmusclewithfluorescentreporters

Fixtheslides

Treatwithreagentfor1hrat37˚C

Imageandcomparetothecontrol

A) Workflow for high throughput screening. B) Absorbance across thewavelengthsfollowthesamepaYernsoanyofthefilterscanbeused.C)Someof the reagents clear differently when used in muscle and in brain. D) Theabsorbanceofthesampleusing405nmlight(right)or620nm(leg).Triplicatesofeachsampleswereused. Ifthenumberis lowerthanPBS,thanit indicatesthat the sample was clearer than the PBS sample. * denotesp<0.05,***denotesp<0.01.ErrorbarsindicateStandarddevia>on.

Compa0bilitywithImmunostaining

Cutthesample

Fixtheslides

Treatwithreagentfor1hrat37˚C

Immunostain

Image

Reagent Sec0onquality Immunostaining

PBS +++ +++Quadrol +++ ++++CUBIC +++ +++

150mMCaffiene +++ ++T-Butyl ++ ++DMSO +++ ++

Reagent GFP TdTomato

PBS +++ +++Quadrol +++ +++CUBIC +++ ++

150mMCaffiene +++ ++T-Butyl +++ ++

DMSO +++ +++

Reagents:JanetRossant,UniversityofToronto(Flk-1+/GFPmice)GabrielleKardon,UniversityofUtah(Pax7Cre)Funding:NIAMS(R01AR062142)(AA)NIAMS(1F30AR066454)(MV)UROP(BSRM)

Name Clearing Technique

Key Component

Time to Clear

Immunostain Morphology

iDISCO Solvent based Dichloromethane/dibenzylether

hours-days yes shrinkage

TDE Simple Immersion

2,2’-thiodiethanol

days-weeks yes same

Sca/e A2 Hyperhydration 4M Urea, 10% glycerol

weeks no expansion

CUBIC Hyperhydration 4M Urea, 50% Sucrose

days yes expansion

PACT Hydrogel Embedding

Histodenz days-weeks yes slight expansion

A) Workflowforobservingtheeffectsofthereagentsonthepreserva>onoffluorescence.B)Qualityofthefluorescenceagerbeingtreatedwiththedifferentreagents.Boththepreserva>onofGFPandTdTomatoweretested.Thescaleforquan>fyingthesamplesis:

++++:thefluorescencequalityisbeYerthanthatofthecontrol(PBS)+++:thefluorescencequalityissimilartothatofthecontrol(PBS).++:thefluorescenceexhibitedbutqualityislessthanofcontrol.+:thefluorescenceisnotexhibited.

Orthogonal view of tdTomato labeled (red) satellite cell and GFP(+) bloodvesselsfromFlk-1+/GFPmice.Schema>crepresenta>onoftheorthogonalviewispresented in the boYom right panel. As seen each view shows a differentnumberofbloodvesselsineachview,representedby(+),(#),(*).Becauseonlyone sec>on can be viewed in tradi>onal analysis, the data obtained can begrossly misrepresented. This scenario highlights the importance of analyzingcellular interac>ons inmuscle >ssue in its na>ve three-dimensional state formoreaccuratequan>fica>onofthisinterac>on.Scalebardenotes25μm.

Dystrophin Merge

PBS

Quadrol

CUBIC

MHCSlow

Dissectandcutthesample

Fixthesample

Treatwithreagentforovernightat37˚C

ImageDeconvolu>on

SNRAnalysis

A B

A B

C

A) Workflowforobservingthecompa>bilityofimmunostainingwiththedifferentreagents.B)TheimagesaretheresultsofimmunolabelingsamplesthathavebeentreatedwithPBS(control),50%QuadrolorCUBIC.ThesampleswereimmunostainedforMyosinHeavyChainSlow(Alexa488),Dystrophin(Alexa568),andDAPI.C)Samplequalityandimmunostainingagerbeingtreatedwiththedifferentreagents.CUBICcocktailshowsdecreaseinimmunostainingquality.Thescaleforquan>fyingthesamplesis:

++++:thequalityisbeYerthanthatofthecontrol(PBS)+++:thequalityissimilartothatofthecontrol(PBS).++:thefluorescenceiss>llexhibitedbutthequalityislessthanthatofthecontrol.+:thefluorescenceisnotexhibited.

Addi>onally,cryosec>onsofthemuscleweretreatedwiththereagentpriortobeingimmunostainedinordertoseeifthetreatmentaffectedi)an>bodysensi>vity/specificityandii)theeffectonbackgroundfluorescence.Ifthe>ssueisabletobestainedviaan>bodies,agerbeingcleared,manyotherreceptorscouldbestainedforinthe>ssue.Thisallowsforthestudyofmanyinterac>onsinthesame>ssue.Addi>onally,theincreaseofbackgroundfluorescencemakesimagingandquan>fica>onofimmunostainingresultsmoredifficultandthereforethereagentshouldn’tincreasebackgroundnoise.

A B

Theworkflowforcremastersampleprep,clearingandanalysis. B) ImagesofFlk-1+/GFPsamples ager being treatedwith the reagent. C) Clearing capabilityandSNRdifferwhen thecremaster sample is treatedwith20%and80%ofareagent.D)Whenusingawidergradient,aclearpeakcanbeobserved(at50%for both of the samples). E)Wider Quadrol gradient images show how 50%Quadrol has greatest signal to noise ra>o. * denotes p<0.05,***denotesp<0.01.ErrorbarsindicateStandarddevia>on.

Flk-1+

/GFP

20%Glycerol 20%RIMSPBS

Dissecthindlimbmuscles

FixthesampleinPFA

Homogenizethesample

Sonicate>ssue

Incubatewithreagentin37°Covernight

Measureabsorp>onoflightusingaplatereader

A B

C

D

405

450

492

620

0.0

0.5

1.0

1.5

2.0

Wavelength(nm)

Abs

orba

nce

Absorbance at various wavelengths show similar trends

PBSUreaTritonQuadrolTriethanolamineDMSOt-Buty alcoholH2OPEGRIMS

PBSUrea

Triton

Quadrol

Triethanolamine

DMSO

t-butynolH2OPEGRIMS

0.0

0.5

1.0

1.5

2.0

96wellPlate.410

Absorbance

Dunnett's multiple comparisons test

PBS vs. UreaPBS vs. TritonPBS vs. QuadrolPBS vs. TriethanolaminePBS vs. DMSOPBS vs. t-butynolPBS vs. H2OPBS vs. PEGPBS vs. RIMS

Mean Diff.

0.30000.29670.57030.5697-0.005667-0.2720-0.3493-0.0850-0.2113

95% CI of diff.

-0.007777 to 0.6078-0.01111 to 0.60440.2626 to 0.87810.2619 to 0.8774-0.3134 to 0.3021-0.5798 to 0.03578-0.6571 to -0.04156-0.3928 to 0.2228-0.5191 to 0.09644

Significant?

NoNoYesYesNoNoYesNoNo

Summary

nsns******nsns*nsns

PBSUrea

Triton

Quadrol

Triethanolamine

DMSO

t-butynolH2OPEGRIMS

0.0

0.2

0.4

0.6

0.8

1.0

96wellPlate.610

Absorbance

Dunnett's multiple comparisons test

PBS vs. UreaPBS vs. TritonPBS vs. QuadrolPBS vs. TriethanolaminePBS vs. DMSOPBS vs. t-butynolPBS vs. H2OPBS vs. PEGPBS vs. RIMS

Mean Diff.

0.23130.16430.34770.40700.02767-0.031000.022000.12730.1277

95% CI of diff.

-0.09179 to 0.5545-0.1588 to 0.48750.02455 to 0.67080.08388 to 0.7301-0.2955 to 0.3508-0.3541 to 0.2921-0.3011 to 0.3451-0.1958 to 0.4505-0.1955 to 0.4508

Significant?

NoNoYesYesNoNoNoNoNo

Summary

nsns***nsnsnsnsns

SNRofclearingreagentsC

PBS

20%

Quad

rol

80%

Quad

rol

20%

Form

amid

e

80%

Form

amid

e

20%

NP40

80%

NP40

20%

Trie

than

olamin

e

80%

Trie

than

lamin

e

20%

Trit

on

80%

Trit

on

20%

Ure

a

80%

Ure

a0

10

20

30

Reagent MIP

Sig

nal t

o N

oise

Rat

io

PBS vs. 20% QuadrolPBS vs. 80% QuadrolPBS vs. 20% FormamidePBS vs. 80% FormamidePBS vs. 20% NP40PBS vs. 80% NP40PBS vs. 20% TriethanolaminePBS vs. 80% TriethanlaminePBS vs. 20% TritonPBS vs. 80% TritonPBS vs. 20% UreaPBS vs. 80% Urea

-8.272-10.113.009-5.080-8.254-9.694-13.48-7.738-8.635-4.302-0.41506.210

-9.843 to -6.702-11.68 to -8.5401.438 to 4.580-6.651 to -3.510-9.825 to -6.684-11.26 to -8.123-15.05 to -11.91-9.309 to -6.167-10.21 to -7.064-5.873 to -2.731-1.986 to 1.1564.639 to 7.780

YesYesYesYesYesYesYesYesYesYesNoYes

***************************************ns****

< 0.0001< 0.00010.0005< 0.0001< 0.0001< 0.0001< 0.0001< 0.0001< 0.0001< 0.00010.5917< 0.0001

Uncorrected Fisher's LSD

20% Quadrol vs. 80% Quadrol20% Formamide vs. 80% Formamide20% NP40 vs. 80% NP4020% Triethanolamine vs. 80% Triethanlamine20% Triton vs. 80% Triton80% Quadrol vs. 20% Urea80% Quadrol vs. 80% Urea

Mean Diff.

-1.838-8.089-1.4405.7444.3339.69516.32

95% CI of diff.

-3.409 to -0.2674-9.660 to -6.519-3.010 to 0.13104.173 to 7.3152.762 to 5.9048.125 to 11.2714.75 to 17.89

Significant?

YesYesNoYesYesYesYes

Summary

*****ns****************

********

****

****

****

********

****

****

****

***

Gradientrevelsop0malconcentra0onD

30% 50% 60% 70% 30% 50% 60% 70% 0

5

10

15

20

25

Sig

nal

to

No

ise

Rat

io

Reagent Zstack

30% vs. 50%30% vs. 60%30% vs. 70%30% vs. 30%30% vs. 50%30% vs. 60%30% vs. 70%

Test details

30% vs. 50%

-6.884-3.074-0.44155.332-3.9462.2882.966

Mean 1

14.73

-11.51 to -2.261-7.696 to 1.549-5.064 to 4.1810.7093 to 9.954-8.569 to 0.6759-2.335 to 6.910-1.656 to 7.589

Mean 2

21.62

YesNoNoYesNoNoNo

Mean Diff.

-6.884

**nsns*nsnsns

SE of diff.

2.180

0.00610.17780.84210.02640.08910.30970.1925

n1

3

Uncorrected Fisher's LSD

30% vs. 50%60% vs. 70%30% vs. 50%60% vs. 70%

Test details

Mean Diff.

-6.8842.632-9.2780.6788

Mean 1

95% CI of diff.

-11.51 to -2.261-1.990 to 7.254-13.90 to -4.656-3.944 to 5.301

Mean 2

Significant?

YesNoYesNo

Mean Diff.

Summary

**ns***ns

SE of diff.

Quadrol Triethaolamine

**

*

30%Quadrol 50%Quadrol 60%Quadrol 70%Quadrol

Flk-1+

/GFP

E

*

#

X

Y

*

#

Z

Y

X

Z

A

C

+

+

B

BA

C

Workflow determining reagents for whole >ssue clearing. Candidates fromeachstagewillallowforthedevelopmentofaclearingcocktailspecificforeach>ssue. This generic process canbe adapted fordifferent>ssues such as theheart,spleenandliver.

A high throughput screen will be used in order to determine the clearingcapabili>es of reagents with the >ssue being observed. Because different>ssueshavedifferentcomposi>on,each>ssuemaymostefficientlyclearwithauniquesetofreagents.Thepurposeofusinghomogenizedmuscleasthefirststep of the experiment is to test clearing efficiency formany reagentswhileusingsmalleramountsof>ssue.Thisscreeningwillallowthetes>ngofalargenumberofreagentsatthesame>me.

20%PEG20%DMSO20%Quadrol

Flk-1+

/GFP

Thehighthroughputscreeningmethodallowsforreagentstobescreenedfortheirclearingcapabili>es.Then,thereagentsareteststoseeintheypreservefluorescence, increase background and allow the applica>on ofimmunohistochemistryoncryosec>ons.Finally,thereagentistestedtoseetheclearing capability, when applied to smaller >ssue. Using this workflow,reagentscanbedeterminedandusedtoclearanytypeof>ssue.

Muscleandbrainresponddifferentlytoclearingagents

Clearingreagentsrespondsimilarlyatvariousspectroscopicwavelengths

Representa0veresultsofscreeningofclearingreagents

405nm 620nm

*** ***

*

***