通(tong)過(guo)不(bu)(bu)同顏色(se)熒光(guang)(guang)(guang)(guang)標(biao)記(ji)不(bu)(bu)同的(de)(de)靶標(biao)，可以(yi)直觀的(de)(de)觀察(cha)同一樣(yang)品細(xi)(xi)(xi)胞內(nei)不(bu)(bu)同結(jie)構(gou)(gou)和(he)蛋(dan)(dan)白(bai)(bai)。熒光(guang)(guang)(guang)(guang)標(biao)記(ji)靶標(biao)的(de)(de)方(fang)法(fa)(fa)主要包括(kuo)熒光(guang)(guang)(guang)(guang)染料、免疫標(biao)記(ji)、熒光(guang)(guang)(guang)(guang)融合蛋(dan)(dan)白(bai)(bai)等——這幾(ji)種(zhong)方(fang)法(fa)(fa)均可選擇性(xing)標(biao)記(ji)細(xi)(xi)(xi)胞內(nei)的(de)(de)結(jie)構(gou)(gou)和(he)蛋(dan)(dan)白(bai)(bai)，讓您(nin)在(zai)成像時更輕松(song)地進行觀察(cha)。細(xi)(xi)(xi)胞生物(wu)(wu)學采用(yong)的(de)(de)很多熒光(guang)(guang)(guang)(guang)工具基(ji)本上(shang)都(dou)是(shi)熒光(guang)(guang)(guang)(guang)基(ji)團。這些(xie)熒光(guang)(guang)(guang)(guang)基(ji)團經(jing)過(guo)不(bu)(bu)同的(de)(de)方(fang)法(fa)(fa)修飾(shi)或結(jie)合到(dao)不(bu)(bu)同的(de)(de)分子上(shang)，從(cong)而具備了(le)某(mou)些(xie)的(de)(de)功(gong)能與(yu)特(te)定的(de)(de)細(xi)(xi)(xi)胞器或蛋(dan)(dan)白(bai)(bai)結(jie)合。通(tong)過(guo)化學修飾(shi)，單個熒光(guang)(guang)(guang)(guang)基(ji)團可以(yi)產生許多變(bian)體形式，且(qie)每種(zhong)變(bian)體都(dou)有不(bu)(bu)同的(de)(de)特(te)異(yi)性(xing)。免疫熒光(guang)(guang)(guang)(guang)細(xi)(xi)(xi)胞化學技術用(yong)于顯示和(he)檢查(cha)細(xi)(xi)(xi)胞或組織內(nei)的(de)(de)抗原或半抗原物(wu)(wu)質。collagenX(COL-10)免疫熒光(guang)(guang)(guang)(guang)試驗(yan)

細胞免(mian)(mian)疫(yi)熒(ying)(ying)(ying)光(guang)實(shi)(shi)驗(yan)注意事項：非(fei)特異性(xing)染(ran)色：是(shi)否滅(mie)活內源(yuan)性(xing)過(guo)(guo)(guo)氧化物酶；孵(fu)育過(guo)(guo)(guo)程中干片(pian)；抗(kang)原熱(re)修復(fu)過(guo)(guo)(guo)度。染(ran)色過(guo)(guo)(guo)深：一(yi)抗(kang)濃度過(guo)(guo)(guo)高；染(ran)色試濃度過(guo)(guo)(guo)高或孵(fu)育時間過(guo)(guo)(guo)長(chang)；染(ran)色劑濃度過(guo)(guo)(guo)高或孵(fu)育時間過(guo)(guo)(guo)長(chang)。通(tong)(tong)常實(shi)(shi)驗(yan)室(shi)先固(gu)(gu)定細胞再進行通(tong)(tong)透(tou)，但若(ruo)檢(jian)測抗(kang)原是(shi)水(shui)不溶(rong)性(xing)蛋白，可(ke)先通(tong)(tong)透(tou)再固(gu)(gu)定，這樣(yang)可(ke)以通(tong)(tong)過(guo)(guo)(guo)通(tong)(tong)透(tou)去除一(yi)些水(shui)溶(rong)性(xing)蛋白，進而(er)(er)可(ke)降(jiang)低(di)免(mian)(mian)疫(yi)熒(ying)(ying)(ying)光(guang)背景(jing)和非(fei)特異性(xing)信號；建議(yi)設陰性(xing)對照組，消除由(you)于抗(kang)體非(fei)特異性(xing)結合而(er)(er)產生的背景(jing)染(ran)色；選擇醛(quan)類(lei)固(gu)(gu)定液時，保持其新鮮度，較(jiao)好(hao)現(xian)配現(xian)用(yong)，使用(yong)不新鮮的醛(quan)類(lei)固(gu)(gu)定液自發熒(ying)(ying)(ying)光(guang)背景(jing)會(hui)升高。collagenX(COL-10)免(mian)(mian)疫(yi)熒(ying)(ying)(ying)光(guang)試驗(yan)免(mian)(mian)疫(yi)熒(ying)(ying)(ying)光(guang)技術利(li)用(yong)熒(ying)(ying)(ying)光(guang)染(ran)料標(biao)記的抗(kang)體與目標(biao)分子結合，從而(er)(er)實(shi)(shi)現(xian)可(ke)視化檢(jian)測。

免(mian)疫熒(ying)(ying)光(guang)(guang)應用范圍(wei)：其應用范圍(wei)極其普遍，可以(yi)測定(ding)內(nei)分泌(mi)、蛋(dan)白質(zhi)、多(duo)肽、核酸、神經遞質(zhi)、受體、細胞因子(zi)、細胞表面抗原(yuan)、肉瘤標志物(wu)、血藥濃度(du)等(deng)各種生(sheng)物(wu)活性物(wu)質(zhi)。根據診斷類別(bie)，又(you)可分為(wei)(wei)傳染性疾病(bing)、內(nei)分泌(mi)、藥物(wu)檢測、免(mian)疫學、血型鑒定(ding)等(deng)。許多(duo)物(wu)質(zhi)都(dou)可產生(sheng)熒(ying)(ying)光(guang)(guang)現(xian)(xian)象，但并非都(dou)可用作熒(ying)(ying)光(guang)(guang)色(se)素。只有(you)那些能產生(sheng)明顯的(de)熒(ying)(ying)光(guang)(guang)并能作為(wei)(wei)染料使用的(de)有(you)機化合物(wu)才能稱(cheng)為(wei)(wei)免(mian)疫熒(ying)(ying)光(guang)(guang)色(se)素或熒(ying)(ying)光(guang)(guang)染料。異硫(liu)氰(qing)酸熒(ying)(ying)光(guang)(guang)素為(wei)(wei)黃(huang)色(se)或橙黃(huang)色(se)結晶粉(fen)末，易(yi)溶于水或酒精等(deng)溶劑。分子(zi)量為(wei)(wei)389.4，較(jiao)大(da)吸(xi)收(shou)光(guang)(guang)波長為(wei)(wei)490495nm，較(jiao)大(da)發射光(guang)(guang)波長520530nm，呈現(xian)(xian)明亮的(de)黃(huang)綠色(se)熒(ying)(ying)光(guang)(guang)。

熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)的(de)(de)產生：一(yi)此(ci)化(hua)學(xue)物質(zhi)能(neng)從外界吸收并(bing)儲存能(neng)量(liang)（如(ru)光(guang)(guang)(guang)(guang)(guang)(guang)能(neng)、化(hua)學(xue)能(neng)等）而進(jin)入激(ji)發(fa)態(tai)，當其從激(ji)發(fa)態(tai)再回復到基態(tai)時，過剩的(de)(de)能(neng)量(liang)可(ke)以(yi)電磁輻射(she)的(de)(de)形(xing)式(shi)放(fang)射(she)（即發(fa)光(guang)(guang)(guang)(guang)(guang)(guang)）。熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)發(fa)射(she)的(de)(de)特點是：可(ke)產生熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)的(de)(de)分子或原子在(zai)接受能(neng)量(liang)后即刻(ke)引(yin)起(qi)(qi)發(fa)光(guang)(guang)(guang)(guang)(guang)(guang)；而一(yi)旦(dan)停止供(gong)能(neng)，發(fa)光(guang)(guang)(guang)(guang)(guang)(guang)（熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)）現(xian)象(xiang)也(ye)隨之(zhi)在(zai)瞬間內(nei)消(xiao)失。可(ke)以(yi)引(yin)起(qi)(qi)發(fa)熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)的(de)(de)能(neng)量(liang)種類很多，由(you)光(guang)(guang)(guang)(guang)(guang)(guang)激(ji)發(fa)所引(yin)起(qi)(qi)的(de)(de)熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)稱為致(zhi)(zhi)熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)。由(you)化(hua)學(xue)應(ying)所引(yin)起(qi)(qi)的(de)(de)稱為化(hua)學(xue)熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)，由(you)X線(xian)或陰極射(she)線(xian)引(yin)起(qi)(qi)的(de)(de)分別稱為X線(xian)熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)或陰極射(she)線(xian)熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)。熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)免疫技(ji)術(shu)一(yi)般應(ying)用致(zhi)(zhi)熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)物質(zhi)進(jin)行標記。免疫熒(ying)(ying)(ying)(ying)光(guang)(guang)(guang)(guang)(guang)(guang)技(ji)術(shu)可(ke)以(yi)用于研究細(xi)胞內(nei)蛋白質(zhi)的(de)(de)定位和表達水平。

免(mian)(mian)疫(yi)熒(ying)(ying)(ying)光(guang)(guang)(guang)間(jian)接法測抗體(ti)(ti)實驗(yan)注(zhu)意事項：1）熒(ying)(ying)(ying)光(guang)(guang)(guang)染(ran)色后一般在1h內完成觀察，或于4℃保(bao)存4h，時(shi)(shi)間(jian)過(guo)長，會使熒(ying)(ying)(ying)光(guang)(guang)(guang)減弱。2）每次(ci)試驗(yan)時(shi)(shi)，需(xu)設置以(yi)下三種對照：①陽性對照：陽性血(xue)(xue)清+熒(ying)(ying)(ying)光(guang)(guang)(guang)標(biao)(biao)(biao)記(ji)物(wu)②陰性對照：陰性血(xue)(xue)清+熒(ying)(ying)(ying)光(guang)(guang)(guang)標(biao)(biao)(biao)記(ji)物(wu)③熒(ying)(ying)(ying)光(guang)(guang)(guang)標(biao)(biao)(biao)記(ji)物(wu)對照：PBS+熒(ying)(ying)(ying)光(guang)(guang)(guang)標(biao)(biao)(biao)記(ji)物(wu)3.已(yi)知抗原(yuan)標(biao)(biao)(biao)本(ben)片需(xu)在操作的(de)各(ge)個步驟中(zhong)，始終保(bao)持(chi)濕潤，避免(mian)(mian)干燥。4.所滴加的(de)待檢抗體(ti)(ti)標(biao)(biao)(biao)本(ben)或熒(ying)(ying)(ying)光(guang)(guang)(guang)標(biao)(biao)(biao)記(ji)物(wu)，應始終保(bao)持(chi)在已(yi)知抗原(yuan)標(biao)(biao)(biao)本(ben)片上，避免(mian)(mian)因放置不(bu)平使液體(ti)(ti)流(liu)失，從(cong)而造成非特異(yi)性熒(ying)(ying)(ying)光(guang)(guang)(guang)染(ran)色。免(mian)(mian)疫(yi)熒(ying)(ying)(ying)光(guang)(guang)(guang)技術(shu)可以(yi)用于研究植物(wu)病理(li)學和農業生產。COL-2免(mian)(mian)疫(yi)熒(ying)(ying)(ying)光(guang)(guang)(guang)染(ran)色

在實(shi)際工作中(zhong)，熒光(guang)抗(kang)原技術應用較少，因此(ci)通常將其稱為熒光(guang)抗(kang)體(ti)技術或免疫熒光(guang)技術。collagenX(COL-10)免疫熒光(guang)試驗

細(xi)(xi)胞(bao)(bao)免(mian)疫熒(ying)光實(shi)驗(yan)(yan)注意事項(xiang)：1、建(jian)議(yi)細(xi)(xi)胞(bao)(bao)直接種孔板，采用倒置(zhi)(zhi)熒(ying)光顯(xian)微鏡拍照(zhao)，這樣可以避免(mian)然(ran)后(hou)(hou)挑片(pian)(pian)(pian)時造(zao)成劃片(pian)(pian)(pian)或(huo)細(xi)(xi)胞(bao)(bao)片(pian)(pian)(pian)破(po)損以及(ji)然(ran)后(hou)(hou)封片(pian)(pian)(pian)可能(neng)造(zao)成滑片(pian)(pian)(pian)等(deng)結果(guo)；2、若(ruo)(ruo)實(shi)驗(yan)(yan)室只有(you)正置(zhi)(zhi)熒(ying)光顯(xian)微鏡，則(ze)需要將(jiang)(jiang)細(xi)(xi)胞(bao)(bao)植于細(xi)(xi)胞(bao)(bao)爬(pa)(pa)片(pian)(pian)(pian)。建(jian)議(yi)直接購買處(chu)理過(guo)的細(xi)(xi)胞(bao)(bao)爬(pa)(pa)片(pian)(pian)(pian)；若(ruo)(ruo)只有(you)普通細(xi)(xi)胞(bao)(bao)爬(pa)(pa)片(pian)(pian)(pian)，可以先將(jiang)(jiang)爬(pa)(pa)片(pian)(pian)(pian)于濃(nong)酸(suan)(suan)(suan)（濃(nong)酸(suan)(suan)(suan)腐蝕性強(qiang)，注意操作）或(huo) 75% 乙(yi)醇浸泡過(guo)夜，若(ruo)(ruo)用酸(suan)(suan)(suan)浸泡過(guo)夜，第二天先用自來水小心(xin)沖洗掉爬(pa)(pa)片(pian)(pian)(pian)殘留的酸(suan)(suan)(suan)液，若(ruo)(ruo)用 75% 乙(yi)醇浸泡，則(ze)不需要此步驟(zou)。然(ran)后(hou)(hou)，用洗潔精(jing)搓(cuo)洗爬(pa)(pa)片(pian)(pian)(pian)，然(ran)后(hou)(hou)用去離子水清洗爬(pa)(pa)片(pian)(pian)(pian)。置(zhi)(zhi)于烘箱 80 ℃ 烘干，再將(jiang)(jiang)爬(pa)(pa)片(pian)(pian)(pian)置(zhi)(zhi)于超凈臺(tai)造(zao)紫外消毒后(hou)(hou)備用。collagenX(COL-10)免(mian)疫熒(ying)光試驗(yan)(yan)

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