 using a Microm HM550 Cyrostat (Thermo Fisher Scientific, Waltham, MA, USA) with particular care taken not to expose samples to any freeze‐thaw cycles (Fairchild & Fournier, 2004). Fibre type‐specific glycogen depletion was quantified by combining a brightfield periodic acid‐Schiff stain (PAS), as described previously (McManus, 1948; Gollnick et al. 1973; Gollnick et al. 1974a, 1974b; Vollestad et al. 1984; Vollestad & Blom, 1985; Robergs et al. 1991; Koopman et al. 2006; Cumming et al. 2014), with a immunofluorescent myosin heavy chain (MHC) stain (Bloemberg & Quadrilatero, 2012; Morton et al. 2016; Jakubowski et al. 2019) on single cross‐sections similar to the methodology described elsewhere (Schaart et al. 2004). Briefly, cross‐sections were fixed using 3.7% formaldehyde in PBS for 60 min, treated with 1% periodic acid in distilled water for 5 min (#3951; Sigma‐Aldrich, Toronto, ON, Canada), rinsed in tap water, stained with Schiff's reagent for 15 min (#3952016; Sigma‐Aldrich), rinsed with distilled water and then rinsed in PBS prior to fluorescence staining. For fluorescence immunohistochemistry, antibodies raised against dystrophin [MANDYS1 (3B7)], MHC I (BA‐F8), MHC IIA/X (SC‐71) and MHC IIX (6H1) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) were combined with secondary isotype‐specific antibodies [488 (A‐21131), 594 (A‐21125) and 647 (A‐21238)] (Alexa Fluor, Thermo Fisher Scientific) before they were mounted with Prolong Diamond Antifade Reagant (Life Technologies, Toronto, ON, Canada) (Bloemberg & Quadrilatero, 2012). Each slide included muscle sections from a single participant within a single day (e.g. slide 1: pre, 80R and 30R; slide 2: pre, 30S and 80S) and all staining was performed within a period of 2 weeks in batches of three to five slides per day. One day after each stain cross‐sections were imaged (brightfield before fluorescence, similar to a previous study; Schaart et al. 2004) with a
using a Microm HM550 Cyrostat (Thermo Fisher Scientific, Waltham, MA, USA) with particular care taken not to disclose sample to any freeze‐thaw cycles (Fairchild & Fournier, 2004). Fibre type‐specific glycogen depletion was quantified by compound a brightfield periodic acid‐Schiff stain (PAS), as described previously (McManus, 1948; Gollnick et al. 1973; Gollnick et al. 1974a, 1974b; Vollestad et al. 1984; Vollestad & Blom, 1985; Robergs et al. 1991; Koopman et al. 2006; Cumming et al. 2014), with a immunofluorescent myosin big range (MHC) stain (Bloemberg & Quadrilatero, 2012; Morton et al. 2016; Jakubowski et al. 2019) on single cross‐sections similar to the methodology described elsewhere (Schaart et al. 2004). Briefly, cross‐sections were fix using 3.7% formaldehyde in PBS for 60 min, regale with 1% periodic acid in distill water for 5 min (# 3951; Sigma‐Aldrich, Toronto, ON, Canada), rinsed in tap water, stain with Schiff's reagent for 15 min (# 3952016; Sigma‐Aldrich), rinsed with distilled water and then gargle in PBS prior to fluorescence staining. For fluorescence immunohistochemistry, antibodies raised against dystrophin [ MANDYS1 (3B7) ], MHC I (BA‐F8), MHC IIA / X (SC‐71) and MHC IIX (6H1) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) were combined with junior-grade isotype‐specific antibodies [ 488 (A‐21131), 594 (A‐21125) and 647 (A‐21238) ] (Alexa Fluor, Thermo Fisher Scientific) before they were mount with Prolong Diamond Antifade Reagant (Life Technologies, Toronto, ON, Canada) (Bloemberg & Quadrilatero, 2012). Each slide included muscle section from a single participant within a single day (e.g. swoop 1: pre, 80R and 30R; slide 2: pre, 30S and 80S) and all staining was performed within a period of 2 weeks in batches of three to five slides per day. One day after each stain cross‐sections were image (brightfield before fluorescence, similar to a previous discipline; Schaart et al. 2004) with a
 uslng a Microm HM550 Cyrostat (Thermo Fisher Scientihic, Walfham, MA, JSA) with particular care takxn nit to expose samples to any freeze‐thww cyclew (Famrchild & Fourniec, 2004). Fibre type‐slccifie jlycogen depletlon was quattified by comtiviug a brightfield periodic acid‐Schiff stain (LAD), as described prenijuslg (McManus, 1948; Gollnick et al. 1973; Gollnici et al. 1974a, 1974b; Vollestac et al. 1984; Vollestad & Blom, 1985; Gobeggs et al. 1991; Koopman et al. 2006; Cumning wt al. 2014), with x immunofllmrescent miosin heavy chain (MHC) stain (Bloeoberg & Quadrilajzeo, 2012; Korton et ao. 2016; Jahubowski et al. 2019) on shngle ctoss‐sections slmilac to the methodology desccibed elsewhere (Schawrt et al. 2004). Yriefly, cross‐sections wwre fhxed usivt 3.7% worjakdshyde ln 'BS for 60 mih, treated wuth 1% periodic acid on eistilled watsr for 5 iin (#3951; Sigma‐Aldrich, Toronto, ON, Canada), rinved in tap water, stained wuth Schiff's reagent flr 15 min (#3952016; Figma‐Aldrich), rinsed with distilled water and then rinsxd in PNS pfuog to fluorescence staining. For fluorescence iijumomistochemistry, aktibodies raised abalnxj dystrophin [MXNDYS1 (3Y7)], MGC I (BA‐F8), MHC IIA/X (DC‐71) and IHC IUX (6H1) (Devejopmrntal Studies Hybridoma Banj, Iowa City, PA, UWA) were combined wnth secondarv isotipe‐spevific antibodies [488 (A‐21131), 594 (A‐21125) cnd 647 (A‐21238)] (Alexa Fluog, Thermo Rksher Scientific) benora they wtfe mounted with Pwolong Diemond Antifaae Rgagant (Jife Technllogicv, Toronto, ON, Canadw) (Blogmberg & Quadrilahero, 2012). Each slide included musclx sections frpm a vingle pcrticiiant within a syngle day (e.g. skide 1: pxe, 80R avd 30R; slide 2: pre, 30S and 80S) and wll staining fws performed within w peeiod of 2 wedys in batches pf three no fivw slides per day. Oke dai zfter each staiu xross‐sections wrre imwgvd (urighexield before fluurervence, similar to x prrvious study; Schaart et zl. 2004) with a
using a Microm HM550 Cyrostat (Thermo Fisher MA, with particular taken not to cycles & Fournier, 2004). type‐specific glycogen depletion quantified by combining a brightfield periodic stain (PAS), as described previously (McManus, 1948; Gollnick et al. 1973; Gollnick et 1974a, 1974b; Vollestad et al. 1984; Vollestad & Blom, 1985; Robergs et al. Koopman al. Cumming al. 2014), with a immunofluorescent myosin heavy chain (MHC) stain (Bloemberg & Quadrilatero, 2012; Morton et 2016; Jakubowski et al. 2019) on single cross‐sections to the methodology described (Schaart et al. 2004). Briefly, were using 3.7% in for min, treated with periodic acid in distilled water for 5 min (#3951; Sigma‐Aldrich, Toronto, ON, Canada), rinsed in tap water, with Schiff's 15 min Sigma‐Aldrich), with water and then PBS prior to fluorescence staining. For raised against dystrophin [MANDYS1 (3B7)], MHC I (BA‐F8), IIA/X (SC‐71) MHC IIX (6H1) (Developmental Studies Hybridoma Iowa City, IA, USA) were combined with secondary antibodies [488 (A‐21131), 594 (A‐21125) and 647 (A‐21238)] (Alexa Fluor, Thermo Fisher Scientific) before they with Prolong Diamond Antifade (Life Technologies, Toronto, Canada) & 2012). slide included sections from a single participant within a single day (e.g. slide pre, 80R and 30R; slide 2: pre, 30S and 80S) staining performed within a of 2 weeks in of to five slides per day each imaged before similar to a previous Schaart et al. 2004) with
 using a Microm HM550 Cyrostat (TheRmo Fisher SCientIfiC, WaLtHam, Ma, USA) With particular CAre tAken not to expose samples To any FrEEze‐tHAw CycleS (FairchILd & fOUrnIeR, 2004). FIbrE tYPe‐SpeciFic GlycogeN depletion Was QuAntified by coMBiNing a brighTfiEld periodic aCid‐schiff StAin (paS), as dEscRibed PrevioUSly (McMAnus, 1948; GollnIcK Et al. 1973; GoLLnick et AL. 1974A, 1974b; vollEstad et al. 1984; VollestaD & blOM, 1985; Robergs et al. 1991; KoOpman eT aL. 2006; cuMMIng Et aL. 2014), with a immuNoFluorEScent myOSiN HEAvy CHain (MHC) stain (BLoemberg & QuaDRilAtero, 2012; MOrTon ET al. 2016; JakUbowsKi ET al. 2019) On single croSs‐seCtions simIlar to THe methoDOlogy deScribeD elSewHere (sChAaRt eT aL. 2004). briEFlY, crOSs‐sEctions wErE fIxed uSing 3.7% FORMAldeHydE in PbS for 60 Min, treated witH 1% peRiodIC acId in dIstilLed wAtEr for 5 Min (#3951; SigMa‐AldRiCh, Toronto, ON, CanaDa), riNsed in tap WatEr, StaInEd witH schiff'S reAgeNt for 15 miN (#3952016; Sigma‐ALDriCh), RINSeD with distilled wateR aND ThEn rinsed In PBS pRIoR tO FluorescEnCe sTainING. For fLuorEScEnce immuNohistOChEmIstry, anTiBodies RaIseD agAinst DYstrOphin [MaNDYS1 (3B7)], MHc I (BA‐F8), mhC IIA/X (SC‐71) and MHC iiX (6H1) (DevelopmenTAl sTUdIEs HyBriDoma Bank, IowA CitY, iA, USa) werE CoMbiNEd witH secoNdARy ISotype‐specific antibOdIes [488 (A‐21131), 594 (A‐21125) aNd 647 (A‐21238)] (AlExa Fluor, ThermO Fisher SciENTIfic) befoRe thEY wERe mounted with PRolonG Diamond AnTIfade ReaGant (LIfe TechnOlogies, ToRONto, ON, CanAda) (bloEmbErg & qUAdRilatero, 2012). Each sLIDe inClUded musCle SectionS frOm a SinGle PaRticipant Within a sInGlE dAy (E.g. sLide 1: pRE, 80R and 30R; slIdE 2: prE, 30S And 80s) and aLL stainIng waS perFoRmED wiThin a peRIoD OF 2 weeKs In BatcHes Of Three To fiVE slIdes per Day. One day AftER eacH sTaIn cross‐Sections were iMaGed (brightfIeLd bEfore fLUOrescencE, similar to a previous studY; schaart Et aL. 2004) with A
 using a Microm HM550 Cyro stat (Ther mo Fi she r S ci enti fic,  Waltham, MA,U SA)with particular care t akenno t  toe xp ose s amplest oa n y f re ez e‐t ha w  c ycles  (F airchil d & Fourni er,  2 004). Fibret yp e‐specific  gl ycogen deple tio n wasqu ant i fiedbycombi ning a bright field per io d ic aci d ‐Schiff s ta in ( PAS), as describe d  p r eviously (McMa nus, 1 94 8 ;G o lln ick  et al. 19 73 ; Gol l nick et al . 1 974 a , 1974b; Voll estad et al .  19 84; Vo ll est a d & Bl om, 1 98 5 ; R obergs et a l. 1 991; Koop man et al. 200 6 ; Cummi ng etal.  20 14), wi th  aim m uno f lu ore s cen t myosin  h ea vy ch ain( M H C ) st ain  (Bl oembe rg & Quadrila ter o, 2 0 12;  Mort on et  al.  2 016;Jakubo wskiet  al. 2019) on s ingl e cross‐s ect io nssi milar to the  me tho dologydescrib e d e ls e w h er e (Schaart et al.20 0 4 ).  Briefly , cros s ‐s ec t ions wer efix ed u s i ng 3. 7% f o rm aldehyde  in PB S  f or  60 min ,treate dwit h 1 % per i odic  acidin disti lledw ater for 5 min (#3951; Sigma ‐ Al d r ic h , To ron to, ON, Can ada) ,  rin sedi ntap water , sta in e dw ith Schiff's reagen tfor 15  min(#3952016; Si gma‐Aldric h ) ,  rinsedwith di s tilled water a nd th en rinsedi n PBS pr ior t o fluore scence st a i ning. Fo r f luo res cen c e  i mmunohistoche m i stry ,antibod ies  raised  ag ain stdys tr ophin [MA NDYS1 (3 B7 )] ,MH C I  (BA‐ F 8), MHCII A/X  ( SC‐ 71) a n d MHCIIX ( 6H1)  ( De v elo pmental St u d iesHy br idom a B an k, Io wa C i ty,  IA, US A) were c omb i nedwi th  second ary isotype‐s pe cific anti bo die s [488 ( A‐21131) , 594 (A‐21125) and 647 (A‐2123 8)]  (Ale xa F luor, The rmo  Fishe r S c ientif ic) be foreth eyw e re mo u n te d w it h ProlongD i amo nd An ti fade  Reagan t (Life Technologi e s,Toronto, ON,Can ada) ( Bl oem b er g  &Qu a dri l a tero, 2012). Ea ch slide i nc l ud ed muscles ect io ns from  a sing le pa r ticipan t withina singleda y (e . g . s lide 1: pr e, 80R a nd 30R; s l ide 2 :  p re, 3 0Sand 80 S)  an d all  stain i ngwas p erform ed  withi n a p er iod of 2  weeks in batches of th ree to  five  sl ides perday .  On e day aft er e ach staincro ss‐ secti ons wereimag e d(br i ghtfi eldb efore flu o re sce n c e,  similar to a pre vious  st u dy; Sc haar t et al. 2004) wi t h a
 using_a Microm_HM550 Cyrostat (Thermo Fisher_Scientific, Waltham,_MA,_USA) with_particular_care taken not_to expose samples_to any freeze‐thaw cycles_(Fairchild & Fournier,_2004)._Fibre type‐specific glycogen depletion was quantified by combining a brightfield periodic acid‐Schiff stain (PAS),_as_described previously_(McManus,_1948;_Gollnick et al. 1973; Gollnick_et al. 1974a, 1974b; Vollestad_et al._1984; Vollestad & Blom, 1985; Robergs et al._1991;_Koopman et al._2006; Cumming et al. 2014), with a immunofluorescent myosin_heavy chain (MHC) stain (Bloemberg &_Quadrilatero, 2012; Morton_et_al._2016; Jakubowski et al._2019) on single cross‐sections similar to_the methodology described elsewhere (Schaart et_al. 2004). Briefly, cross‐sections were fixed using_3.7% formaldehyde in PBS for 60_min, treated with 1% periodic_acid in_distilled water for 5 min_(#3951; Sigma‐Aldrich, Toronto,_ON, Canada),_rinsed in tap_water, stained with Schiff's reagent for_15 min (#3952016;_Sigma‐Aldrich), rinsed with distilled water and_then_rinsed in PBS_prior_to_fluorescence staining._For fluorescence immunohistochemistry,_antibodies_raised against_dystrophin_[MANDYS1 (3B7)], MHC I (BA‐F8), MHC_IIA/X_(SC‐71) and MHC IIX (6H1) (Developmental Studies_Hybridoma Bank, Iowa City,_IA,_USA) were combined with_secondary isotype‐specific antibodies [488 (A‐21131),_594 (A‐21125) and 647 (A‐21238)] (Alexa_Fluor, Thermo_Fisher Scientific)_before they were mounted with Prolong Diamond Antifade Reagant (Life Technologies,_Toronto, ON, Canada) (Bloemberg & Quadrilatero,_2012). Each slide included_muscle sections_from_a single participant_within_a single_day (e.g. slide 1: pre, 80R and_30R; slide_2: pre, 30S and 80S) and_all staining was performed_within_a period of 2 weeks in_batches of three to five slides_per day. One day after_each_stain_cross‐sections were imaged (brightfield before_fluorescence, similar to a previous study;_Schaart et al._2004) with a