1、T e : Z Z , o , 1982 M 9 3 , 2 C , V 3 b Z _ h b “ : 15006489735; Enullmai:l doufangnullfang2009 126. comAbout DOU FangnullFang: Female,born in September, 1982. Postgraduatestudent. Te:l 15006489735; Enullmai:ldoufangfang2009 126. coml : 2010null03null30 : 2010null05null18 I : null “ : S E 1 S “ (I
2、| : 30772386); 8 1 S “ (I | :Y2006C94)T : 266071null 8 g , v D ( Z Z ); 266071 8 g , 8 S , 8 S L i null 8 y S E L i ( nullS , k ; )Y T : k ; , Enullmai:l dxgdoctor hotmai.l comReceiveddate:M ar30, 2010Accepted date:M ay 18, 2010Foundation item: National NaturalScience Foundation of China ( No:307723
3、86); Natural Science Foundanulltion of Shandong Province ( No:Y2006C94)From theMedical College of QingdaoUniversity(DOU FangnullFang), Qingdao266071, Shandong Province, China;the State Key Laboratory CultivationBase, Shandong ProvincialKey Labonullratory of Ophthalmology, ShandongEye Institute(LIU T
4、 ing, DONG XiaonullGuang), Qingdao 266071, ShandongProvince, ChinaResponsible author: DONG XiaonullGuang, Enullmai:l dxgdoctor hotmai.lcomL !l j f ; / ) null窦方方null 刘null 廷null 董晓光Immunofluorescence staining techniques forretinal stretched preparation inmiceDOU FangnullFang, LIU Ting,DONG XiaonullGu
5、angKeywords!null retina stretched preparation; immunofluorescence; eyeball ofmouseAbstract!null Objectivenull To search for a simple and easily handled immunofluoresnullcence technique for retinal stretched preparation in m ice. Methodsnull A total of 30C57BL/6J adult m ice were perfused w ith 40 g
6、L- 1 paraformaldehyde through theheart. Eyeballswere enucleated and retinaswere removed. A llm ice were divided rannulldom ly into 3 groups. Twenty retinas in groupA w ere treatedw ith3 g L- 1 TritonXnull100at4 # for 1 hour; Twenty retinas in group B were firstly put intomethanol for 30m inullnutes,
7、 and then treatedw ith 3 g L- 1Triton Xnull100 at4 # for 1 hour; Twenty retinas ingroup C w ere firstly put intomethanol for 30 minutes, and then treated w ith 3 g L- 1TritonXnull100 for staying overnightat4# ; anullsmoothmuscle actin (anullSMA) immunofluonullrescence stainingwas used, and photograp
8、h was taken under fluorescence microscopein all groups. The differencew as observed. Resultsnull Under fluorescencem icroscope,morphologies of retinal vesselswerew ith weak fluorescence and fuzzy circumscriptionin group A; Retinas were w ith fluorescence, vascular branches were seen clearly butw ith
9、weak fluorescence in groupB; Retinaswere w ith strong fluorescence, anullSMA w asfound in cellmembranesofvascularendothelialcellsand perithelial cells, w ith vacuolusand light background staining. Conclusionnull Retinal stretched preparation immunoflunullorescence is a better, simple and reliable w
10、ay to observe morphologies of retinal tisnullsues. In the experiment, the key for successful staining is to defatw ithmethanol andsoakw ith enoughTritonXnull100.RecAdvOphthalmol2010;30(8): 714null717 m s | !null R774. 1null null D S !null A c I | !null 1003null5141( 2010)08null0713null041 o M !null
11、视网膜铺片;免疫荧光;小鼠眼球K 1 !null “ null 探讨一种简便a易操作的小鼠视网膜铺片免疫荧光染色技术bZ E null 30只 C57BL/6J成年小鼠行 40 g L- 1多聚甲醛心脏灌注固定,摘取眼球,剥离视网膜,随机分为 3组: A组 20个视网膜直接置于 3 g L- 1TritonXnull100中 4# 孵育 1 h; B组 20个视网膜先置入甲醇冰上固定 30 m in,再置于 3 g L- 1Triton Xnull100中 4# 孵育 1 h; C组 20个视网膜先置入甲醇冰上固定 30 m in,再置于 3 g L-1Triton Xnull100中 4 #
12、 孵育过夜b各组分别行nullnull平滑肌肌动蛋白免疫荧光染色,荧光显微镜下照相,观察其差异b T null 荧光显微镜下观察, A组视网膜血管荧光微弱,血管模糊不清; B组视网膜可见荧光,血管分枝清晰可见,但荧光较弱; C组视网膜荧光强亮, nullnull平滑肌肌动蛋白特异性表达在血管内皮细胞及周细胞的胞膜上,荧光呈空泡状,背景染色轻b null 视网膜铺片免疫荧光染色是一种较好的a简便易行的视网膜组织形态学观察方法,实验过程中甲醇脱脂处理和足够的 TritonXnull100浸泡是保证染色成功的关键b S Z null 2010; 30(8): 714null717“ - j F 4
13、Z E F M 1a9 2# h 5 3 ,9 v 7 ; I S : Z E j j % 4 4b j 5 / T N aZ 4 v ah H W g y , 4 ! dB j 5 5b j f ;/ S = n D ,8 S D 6null8 L l , g j f ; / ,C / b714 null http: / /www. ykxjz. com S Z null 2010 M 8 null 30 null 8 RecAdvOphthalmolnull Vol. 30No. 8August20101null Z E1. 1null 1. 1. 1 L null C57BL/6J M
14、l 30 , Y K , b ) , 1 Z X L , ! Hq + h 8 L S b1. 1. 2null L k 4 null F nullnull ( alphanullsmoothmuscle actin, nullnullSMA) X H F 8 a F IgGnulls ; e ; ( fluorescein isothiocyanate,FITC)a b 1 q p V 3 , Y = ? A (N ikon Eclipase E800, ), S g al g a g a , g aA Z 0 aA Z 0 ( D K ), 1. 5 mL EP5 , A # 20 mL
15、b1. 2null Z E1. 2. 1null 9 % null | 30 C57BL/6J M l , o (0. 1 g kg- 18 )a o d ( 0. 125g kg- 18 ) 8 = J b A , 2 A s Y 3 40 g L- 1 J A b % l ,P m Z 0 C t l / , m g g B l g ,_ | g / , s / F , , L _ g 7 , , | , i J _ s # b U P 2mm) 7| A h O i g 7 9 , 9 3 100mL, M V 9 , A 7 1 , 9 40g L- 1 J A 100mL,l V 9
16、 % A , T a null | 60 o 9 3 ! , m A / g o g 7 ,“ a a 8 , : / j ,b M 7 4 , Z C o , j j , “ R , * _ Z , , Ch ? Z | j 8 8 G , “ 8 F PBS, ! b j s 3F : AF 20 j 3 g L- 1Triton Xnull100 4 # 1 h; BF 20 j 5 J ? b % 30 min, 3 g L- 1 Triton Xnull100 4 # 1 h; CF 20 j 5 J ? b % 30min, 3 g L- 1 Triton Xnull100 4 #
17、 V b F j V Triton Xnull100 | F b i F 2 h, A , ! b Q anullSMA X H F 8 ( 1: 100, 1 g L- 1Triton Xnull100 d ) 37 # 3 h, H ! | v , F B F , PBS 9 b ( 5 1 g L- 1 TritonXnull100! 3Q , Q 20m inb F F IgGnullFITC( 1: 100, 1 g L- 1 Triton Xnull100 d ) i E ; 2h, ( 5 1 g L- 1 Triton Xnull100! 3Q , Q 20m inb m A
18、/ , * _ , F ; b 4 , ; A 490 nmo / 4 iv M b2null T2. 1null v 8 4 null l # 9 % , | oH o F A = ,V 9 ; j “ b , ,; i , A A b2. 2null ; A / 4 null ; A / 4 , AF / j 5 ; ,M ; , “ ; ! % T ,9 T 9 h b(2) % H W 1 g z , J a o v l M M 1 , , o v ,% H W V a 2b % H W V , V n j = L , Y 4 (m 4); % HW V , = , j c , j 7
19、 (m 5); % 25min, j = , 716 null http: / /www. ykxjz. com S Z null 2010 M 8 null 30 null 8 RecAdvOphthalmolnull Vol. 30No. 8August2010 (m 6)b( 3) j 5 b M 7 , T H M 7 , L V j w ,! ,K L = b(4) j s j 8 A 8 s 1 , : / 8 , C h ? G “ , T j 88 = / ; null S , , k ; . l j F F M T Z E ) J. S Z , 2009, 29(3): 16
20、1null164.2null + , null S , k ; .l 5 9 / j T / #/ F ) J. S Z , 2009, 29(9): 663null666.3null a , l , bo , r : , * ,f null . j h f ; / j 5 % h J. S , 2003, 3(7): 977null979.4null ,f M , . v j % I S : Z E y J. S Z , 2004, 24(5): 341null344.5null W , null ,f null , , . j 5 / # U h j h J.S = S , 2005, 5
21、( 6): 1139null1141.6null Huang Q, W ang S, Sorenson CM, SheibaniN. PEDFnulldeficientm iceexhibit an enhanced rate of retinal vascular expansion and aremore sensitive to hyperoxianullmediated vessel obliteration J. ExpEyeRes, 2008, 87(3): 226null241.7null Zeisberg EM, Potenta S, Xie L, Zeisberg M, Ka
22、lluriR. Discovery ofendothelial tomesenchymal transition as a source for carcinomanullassociated fibroblastsJ. Can cer Res, 2007, 67(21): 10123null10128.8null Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A,Abramsson A, et al. VEGF guides angiogenic sprouting utilizing ennulldothelial tip cell filopodia J. J Cell Biol, 2003, 161( 23): 1163null1177.9null null w , , k ; , null , null . Evans 9 5 / l j 3 5 4 J. S Z ,2007, 27(4): 258null261.717 S Z null 2010 M 8 null 30 null 8 RecAdvOphthalmolnull Vol. 30No. 8August2010 http: / /www. ykxjz. com
