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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
*** ***
*
***
