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Reproducibility
Cell selleck loss
9
8
7
6
5
1.08
1.04
1.00
0.96
0.92
0.88
100
80
60
40
20
0
Cycle
1
2
3
4
5
Alexa-488
Alexa-555
Alexa-647
0
ten
twenty
thirty
forty
0
0.01 0.03 0.1 0.3
1
1
2
3
4
5
Time exposed (min)
Vemurafenib concentration (?M)
CycIF cycle
Figure1|Multiplexedimagingofsingle-cellusingCyclicImmunoFluorescence(CycIF).(a)AnoverviewoftheCycIFprocedure.Four-colourstaining
alternateswith?uorophoreinactivationbyoxidationtoprogressivelybuildamultichannelimage.(b)CycIFprocedureusingdirectimmuno?uorescence
(with?uorophore-conjugatedantibodies)andchemicalinactivationof?uorophores.COLO858melanomacellswere?xedandstainedusingantibodiesfor
Ki-67(Alexa488),p-HistoneH3(Alexa555),p21(Alexa647)andHoechst(leftpanel).

Cellswereexposedto?uorophore-inactivationbyoxidationusing
hydrogenperoxide,highpHandlightandthenreimaged(middlepanel)tocon?rmef?cientbleaching.Cellswerethenstainedwith?uorophore-conjugated
antibodiesforp-S6S240/244(Alexa488),p-RbS807/811(Alexa555),p-S6S235/236(Alexa647)andHoechst.(c)CycIFprocedureusingindirect
immuno?uorescenceandprotease-mediatedantibodystripping.MCF7cellswere?xedandstainedusingprimaryantibodiesforp-ERK1/2T202/Y204
(rabbit),p53(mouse),Alexa488-conjugatedanti-rabbit,andAlexa647-conjugatedanti-mousesecondaryantibodies(leftpanel).Cellsweredigestedwith
pepsin/papainmixture(seeMethodsfordetails)andreimaged(middlepanel).Cellswererestainedusingprimaryantibodiesforp-RbS807/811
(rabbit),
p-HistoneH2A.XS139(mouse),Alexa488-conjugatedanti-rabbit,andAlexa647-conjugatedanti-mousesecondaryantibodies(rightpanel).Minoxidil >
(d )Bleaching
rateforAlexa488,555or647-conjugatedantibodiesfollowingincubationinabase-hydrogenperoxidemixture.(e)Correlationofsignalintensitiesafter
usingthesameantibodiesinsuccessiveCycIFcycles.Five-cycleCycIFwasappliedtoCOLO858cellstreatedwithincreasingdosesofvemurafenib(error
barsshowtherangeofbiologicalduplicates).CellswerestainedwithAlexa488-conjugatedp-ERKantibody,andp-ERKsignalintensitiesfromdifferent
CycIFroundswerequanti?edandcompared.Cells(1,000�C2,000)wereimagedforeachconditionandwellmeanintensityvaluesacrossduplicateswere
reported.Errorbarsindicates.d.(f)Quanti?cationofcelllossbasedonHoechststaining(averagedacrossn?30differentwells)throughcyclesofCycIF.


CellnumbersfromeachwellaftereachCycIFcyclewerenormalizedtothemeancellnumberderivedfromcycle1andpresentedinbox-and-whiskerplots
withmeanvalues(shownbyredlines),interquartileranges(shownasboxes)andwhiskers(representingthe1st/99thpercentiles).
conveRegorafenib BAY 73-4506 ntional IF procedures. Additionally, the fraction of cells lost detached inthe NaOH-H2O2 mixture, and 0�C2% per cycle sub-
throughcyclesofwashingandimagingislow,approximately2�C sequently(Fig.1f).Lowcelllossispartlyaconsequenceofouruse
5% in the ?rst and second cycles, during which adherent cells ofplatewashersforwashingandstainingsteps(seeMethods)and
NATURE COMMUNICATIONS |6:8390|DOI: ten.1038/ncomms9390 |www.nature.com/naturecommunications
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ARTICLE
NATURECOMMUNICATIONS|DOI:ten.1038/ncomms9390
paraformaldehyde ?xation; the use of methanol ?xation alone Combin