1.过氧化氢和超氧阴离子染色(DAB和NBT染色)

过氧化氢和超氧阴离子染色（DAB/NBT染色）取样，用打孔器选择叶片深绿，长势较好的叶片打孔，避免接触主次叶脉，叶片的中上部位最佳，取10个叶圆片于培养板内。暗中加入2mlDAB（NBT）/孔，盖盖，用锡箔纸包好（避光）28口，6h（lh）。90%乙醇70□沸水浴脱色。保存在50%甘油中。400倍放大观察H2O2（02.-）的含量及位置°（BX61,奥林巴斯）DAB溶液配置：1mg/mL二甲基联苯胺DAB反应液中（pH5.5，50mMTris-HCl）现配现用。l.97gTris-HCl定容到250ml用HCl调至5.5+0.25gDABNBT溶液配置：0.5mg/mL氮蓝四磋NBT反应液中（pH7.8，50mM磷酸缓冲液）现配现用。PPS缓冲液，A91.5ml+B8.5ml稀释到400ml+2gNBT参考文章-i-DMTU斗巴q-i-DMTU斗巴qGrowrhinNomwajscuiii'il慕ion：phct<K5ynitih«Fj5jedglutJithicinE-miax[homeost-aEiEmiimiftuvncedbyHytl罪巒门irtHauLlXueMinWdru.*1.UCbcn/*.GulimJiJalAhmmE-d-1'.ZojiiShi11.MIeHmOJ.Can■IJdngQ^JinYuUnklar^fZhau*-"Growthremperarure-inducedchangesinbiomassaccumulation,photosynthesisandglutathioneredoxhomeostasisasinfluencedbyhydrogenperoxideincucumberHaoLi：XueMinWK学：Li匚hena,GolamJalalAtiammed3,XiaoJianXiaa,KaiShiapMichdelJ.ConsidinertJingQuanYuah4YdrtHongZhuun"4J.LipidperoxrdatronanalysisendH2O2accumulationassayLipidperoxidationinleaveswasestimatedbymeasuringthemalondialdehyde(MDA)productionbythethiobarbituricacid(TBA)methoddescribedbyRef[33|.H2O2wasextractedfromEeaftissueandthenanalyzedaccordingtoRef.[34].Forthehisto・chemicalstainingofH2O2inplants,leavesweredetachedandplacedinasohitioncontaining1mgmL13.3^dtarnincbenzidine(DAB.pH5.5)for6hafterali^ghtvacuuminfiltration.Leafdiscswereboiledin95%(v/v)ethanolfor10min.storedinSOXglycerolandthenphotographedunderanOlympusmotorize