Zeiss ELYRA - Post-fixation and Imaging Buffer for dSTORM; Post-fixation for Antibody Labeling; Imaging Buffer Preparation for dSTORM

Post-fixation Following Antibody Labeling
A post-fixation step can be valuable. Herein, you fix cells a second time after staining in order to improve the stability
ofthelabel.Thiscanpreventthelabelfromdetachingandoatingtheimagingmedium.
Imaging Buffer dSTORM
(pleasesee:G.T.Dempsey,NatureMeth2011andS.vanLinde,NatureMeth2011)Itisrecommendedtofreshly
preparetheimagingbuffereveryday.Theoxygenscavengersystemwillonlylastforafewhoursandismainlyneeded
forCyaninedyeslikeAlexa647andCy5(RhodaminesandOxazinesdonotrequireit.)
Imaging Buffer
100µlPBS10x(Phosphatebufferedsaline;e.g.fromSigma:D1408)
100µlMEA(CysteamineHydrochloride)stockconcentration1M(e.g.fromSigmaM6500-25G)(toxic!!)
Optional:
Oxygenscavengersystem(forcyaninedyes)
500µlGlucose20%(e.g.insolutionfromSigma(49163-100ML))
25µlGlucoseOxidase(e.g.24mg/mlGluOxfromAspergillusniger;SigmaG0543-50KU)
5µlCatalase(e.g.:12.6mg/mlCatalasefromBovineliver;SigmaC3155-50MG)
AddH2Otonalvolumeof1ml
Veryimportant:adjustpHto7.5-8.5with5MNaOHor4.5MKOH
(TotestpHyoucanusepHpaperintheindicatedrange)
Note
1MMEA(SigmaM6500-25G):0,113gin1ml
StoreMEA(solid)at4ºC.Preparefreshas1Mworkingstocksolutioninwater.Thisstockcanbekeptat4ºCand
usedwithin1-2weeksofpreparation.Alternativelyyoucanfreezesmallaliquotsat-20ºCandkeepthemforseveral
months.
TheMEAconcentrationdependshighlyontheuorophoreandtheexperimentalcondition.Thereforethebest
concentration has to be tested by trial and error for each sample (between 10mM and 100mM).
IfyouuseCysteamine(nottheHydrochloride)youhavetouse37%HCltoadjustthepHto7.5-8.5
As a general advice:
• Ifblinkingratesaretoohigh:increaseMEAconcentrationand/orincreasepH
• Ifblinkingratesaretoolow:decreaseMEAconcentrationand/ordecreasepH
White Paper
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