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SILENCING$BETA2$ADRENERGIC$RECEPTORS…$$

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After! several! washes! in! PBS! and! preYincubation! in! PBS! with! 3%! blocking!

serum! for! 1! h,! cells! were! incubated! with! the! primary! goat! polyclonal! antiYbeta2!
(Ab40834! from! abcam)! diluted! 1:100.! After! this! incubation,! cells! were! washed!
with! PBS! and! incubated! in! a! dark! chamber! with! the! secondary! antibody! donkey!
antiYgoat! IgGYFITC! at! 1:150! (Jackson! ImmunoResearch,! PA,! USA)! for! 1! h! to! 37ºC.!
Then,!the!samples!were!observed!under!a!confocal!microscope!(Axiovert!200;!Carl!
Zeiss! Meditec! GmbH,! Jena,! Germany),! equipped! with! a! PASCAL! confocal! module!
(LSM!5;!Zeiss).!All!images!were!managed!with!the!accompanying!PASCAL!software.!

Eyes! were! fixed! in! 4%! paraformaldehyde! in! PBS! 1X! overnight! at! 4ºC.! Eyes!
were!washed!twice!in!PBS!1X!for!15!min,!and!immersed!in!11%!sucrose!overnight!
at!4ºC.!The!following!day!the!eyes!were!immersed!in!33%!sucrose!prepared!in!PBS!
1X.!Finally,!the!eyes!were!embedded!in!OCT!(Leica).!5Y10!µm!sections!were!made!
using!a!LEICA!CM!1850!cryotome,!in!3Yaminopropiltrietoxisilane!coated!glasses.!

HaematoxylinYeosin! staining! was! performed! using! the! following! protocol:!
Sections! were! fixed! and! washed! in! water! for! 5! minutes.! Haematoxylin! (Carazzi´s!
Heamtoxylin,! Panreac,! Barcelona,! Spain)! was! incubated! for! 1! minute! and! sections!
were! washed! in! water! for! 10! minutes.! Eosin! (Eosin! Bluish,! Panreac)! contrast! was!
made!and!finally!sections!were!washed!serially!with!96º!and!100º!ethanol.!Finally,!
xilol! treatment! was! performed! to! dehydrate! the! sections! previous! to! their!
observation!by!means!of!the!microscope.!

Microscopy!was!performed!with!a!confocal!Zeiss!Axiovert!200M!microscope!
(Zeiss,!Jena,!Germany),!equipped!with!a!LSM!5!Pascal!confocal!module.!All!images!
were!managed!with!the!LSM!5!Pascal!software.!

2.4.$Side$effects$

The! assessment! of! the! ocular! tolerance! of! the! RNAi! sequences,! were!
followed! several! aspects! and! parameters! from! the! tear! film,! ocular! anexa! and!
ocular! surface.! The! analysis! were! performed! and! graded! before! and! after! the!
topical! application! of! siRNA! with! the! Efron! grading! scales.! A! Topcon! SLY8Z! slit!
lamp!and!a!Topcon!SPY2000P!specular!microscope!(Topcon!Spain,!Madrid,!Spain),!
as! well! as! a! digital! camera! and! a! “de! visu”! exam,! were! used! for! every! experiment.!
SlitYlamp! images! were! captured! under! 10X! augments,! a! diffuser! filter! and! a!
medium! degree! of! illumination.! The! images! were! recorded! and! analysed! by!
IMAGEnet!2000!system!software!(Topcon).!

2.5.$Data$analysis$

All! data! are! presented! as! the! mean+s.e.m.! Significant! differences! were!
determined! by! twoYtailed! Student's! tYtests.! The! plotting! and! fitting! of! all! the! data!
was!carried!out!with!the!computer!programme!Prism!GraphPad!v.4.0!(Prism,!USA).!!

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