通過不(bu)同(tong)顏色熒(ying)光(guang)(guang)(guang)標記(ji)不(bu)同(tong)的(de)靶(ba)標，可(ke)以(yi)直觀(guan)(guan)(guan)的(de)觀(guan)(guan)(guan)察(cha)同(tong)一樣品細(xi)胞(bao)內不(bu)同(tong)結(jie)構和蛋(dan)白。熒(ying)光(guang)(guang)(guang)標記(ji)靶(ba)標的(de)方(fang)法主要包括熒(ying)光(guang)(guang)(guang)染料、免疫(yi)標記(ji)、熒(ying)光(guang)(guang)(guang)融合(he)蛋(dan)白等——這幾種(zhong)方(fang)法均(jun)可(ke)選擇性(xing)標記(ji)細(xi)胞(bao)內的(de)結(jie)構和蛋(dan)白，讓您(nin)在成像時更輕松(song)地(di)進(jin)行觀(guan)(guan)(guan)察(cha)。細(xi)胞(bao)生物(wu)學采用(yong)的(de)很多熒(ying)光(guang)(guang)(guang)工具(ju)(ju)基(ji)(ji)本上(shang)都是(shi)熒(ying)光(guang)(guang)(guang)基(ji)(ji)團。這些(xie)熒(ying)光(guang)(guang)(guang)基(ji)(ji)團經(jing)過不(bu)同(tong)的(de)方(fang)法修(xiu)飾(shi)或(huo)結(jie)合(he)到不(bu)同(tong)的(de)分(fen)子(zi)上(shang)，從而具(ju)(ju)備了某些(xie)的(de)功能(neng)與(yu)特定的(de)細(xi)胞(bao)器(qi)或(huo)蛋(dan)白結(jie)合(he)。通過化學修(xiu)飾(shi)，單個熒(ying)光(guang)(guang)(guang)基(ji)(ji)團可(ke)以(yi)產(chan)生許多變體形(xing)式(shi)，且每種(zhong)變體都有不(bu)同(tong)的(de)特異性(xing)。免疫(yi)熒(ying)光(guang)(guang)(guang)細(xi)胞(bao)化學技(ji)術用(yong)于顯示和檢查細(xi)胞(bao)或(huo)組織內的(de)抗原或(huo)半抗原物(wu)質(zhi)。collagenX(COL-10)免疫(yi)熒(ying)光(guang)(guang)(guang)試驗

細胞免(mian)疫熒光(guang)(guang)實(shi)驗(yan)(yan)注意事項：非(fei)特(te)異性(xing)(xing)染(ran)(ran)色：是否滅活內源性(xing)(xing)過(guo)氧化物(wu)酶；孵(fu)育(yu)過(guo)程中干片；抗(kang)(kang)原熱(re)修復過(guo)度(du)(du)。染(ran)(ran)色過(guo)深：一抗(kang)(kang)濃(nong)度(du)(du)過(guo)高；染(ran)(ran)色試濃(nong)度(du)(du)過(guo)高或孵(fu)育(yu)時間過(guo)長；染(ran)(ran)色劑濃(nong)度(du)(du)過(guo)高或孵(fu)育(yu)時間過(guo)長。通(tong)(tong)(tong)常實(shi)驗(yan)(yan)室先固(gu)定(ding)細胞再(zai)進行(xing)通(tong)(tong)(tong)透，但(dan)若(ruo)檢測抗(kang)(kang)原是水不溶性(xing)(xing)蛋白，可先通(tong)(tong)(tong)透再(zai)固(gu)定(ding)，這樣可以通(tong)(tong)(tong)過(guo)通(tong)(tong)(tong)透去除一些水溶性(xing)(xing)蛋白，進而可降低(di)免(mian)疫熒光(guang)(guang)背(bei)(bei)景(jing)和非(fei)特(te)異性(xing)(xing)信號(hao)；建議設陰性(xing)(xing)對照(zhao)組(zu)，消除由(you)于抗(kang)(kang)體非(fei)特(te)異性(xing)(xing)結合(he)而產生的(de)背(bei)(bei)景(jing)染(ran)(ran)色；選擇(ze)醛類(lei)固(gu)定(ding)液時，保持(chi)其新鮮度(du)(du)，較好現(xian)配現(xian)用，使用不新鮮的(de)醛類(lei)固(gu)定(ding)液自(zi)發熒光(guang)(guang)背(bei)(bei)景(jing)會升(sheng)高。collagenX(COL-10)免(mian)疫熒光(guang)(guang)試驗(yan)(yan)免(mian)疫熒光(guang)(guang)技術利用熒光(guang)(guang)染(ran)(ran)料標記的(de)抗(kang)(kang)體與目標分(fen)子結合(he)，從而實(shi)現(xian)可視化檢測。

免疫熒(ying)(ying)光(guang)(guang)(guang)(guang)(guang)應用范圍(wei)：其應用范圍(wei)極其普遍，可(ke)(ke)以(yi)測定(ding)內(nei)分(fen)泌(mi)、蛋白質、多肽、核(he)酸、神經(jing)遞(di)質、受(shou)體、細胞因(yin)子(zi)、細胞表(biao)面抗原、肉瘤標志物、血藥濃度等各種(zhong)生物活性物質。根據診(zhen)斷類別，又可(ke)(ke)分(fen)為傳染性疾(ji)病、內(nei)分(fen)泌(mi)、藥物檢測、免疫學、血型鑒(jian)定(ding)等。許多物質都(dou)可(ke)(ke)產生熒(ying)(ying)光(guang)(guang)(guang)(guang)(guang)現(xian)象，但并(bing)非都(dou)可(ke)(ke)用作熒(ying)(ying)光(guang)(guang)(guang)(guang)(guang)色(se)素。只有那些能(neng)產生明顯的(de)熒(ying)(ying)光(guang)(guang)(guang)(guang)(guang)并(bing)能(neng)作為染料(liao)使用的(de)有機(ji)化合物才能(neng)稱為免疫熒(ying)(ying)光(guang)(guang)(guang)(guang)(guang)色(se)素或(huo)熒(ying)(ying)光(guang)(guang)(guang)(guang)(guang)染料(liao)。異硫氰酸熒(ying)(ying)光(guang)(guang)(guang)(guang)(guang)素為黃色(se)或(huo)橙黃色(se)結(jie)晶粉末(mo)，易溶(rong)于(yu)水或(huo)酒精(jing)等溶(rong)劑。分(fen)子(zi)量為389.4，較大吸收光(guang)(guang)(guang)(guang)(guang)波(bo)長(chang)為490495nm，較大發射光(guang)(guang)(guang)(guang)(guang)波(bo)長(chang)520530nm，呈現(xian)明亮(liang)的(de)黃綠色(se)熒(ying)(ying)光(guang)(guang)(guang)(guang)(guang)。

熒光(guang)(guang)(guang)的產(chan)生(sheng)：一(yi)此化(hua)(hua)學(xue)(xue)物質能(neng)(neng)從外(wai)界吸收并儲存能(neng)(neng)量(liang)（如光(guang)(guang)(guang)能(neng)(neng)、化(hua)(hua)學(xue)(xue)能(neng)(neng)等）而進(jin)入激發(fa)(fa)態，當其從激發(fa)(fa)態再回復到基態時，過剩的能(neng)(neng)量(liang)可以(yi)電磁輻射(she)的形(xing)式(shi)放射(she)（即(ji)(ji)發(fa)(fa)光(guang)(guang)(guang)）。熒光(guang)(guang)(guang)發(fa)(fa)射(she)的特點是：可產(chan)生(sheng)熒光(guang)(guang)(guang)的分子或原子在接(jie)受能(neng)(neng)量(liang)后即(ji)(ji)刻引(yin)起(qi)(qi)(qi)發(fa)(fa)光(guang)(guang)(guang)；而一(yi)旦停止供能(neng)(neng)，發(fa)(fa)光(guang)(guang)(guang)（熒光(guang)(guang)(guang)）現象(xiang)也隨之在瞬間內消(xiao)失。可以(yi)引(yin)起(qi)(qi)(qi)發(fa)(fa)熒光(guang)(guang)(guang)的能(neng)(neng)量(liang)種類很多，由(you)光(guang)(guang)(guang)激發(fa)(fa)所引(yin)起(qi)(qi)(qi)的熒光(guang)(guang)(guang)稱為(wei)致(zhi)熒光(guang)(guang)(guang)。由(you)化(hua)(hua)學(xue)(xue)應所引(yin)起(qi)(qi)(qi)的稱為(wei)化(hua)(hua)學(xue)(xue)熒光(guang)(guang)(guang)，由(you)X線(xian)或陰(yin)極射(she)線(xian)引(yin)起(qi)(qi)(qi)的分別稱為(wei)X線(xian)熒光(guang)(guang)(guang)或陰(yin)極射(she)線(xian)熒光(guang)(guang)(guang)。熒光(guang)(guang)(guang)免疫技(ji)術(shu)(shu)一(yi)般應用致(zhi)熒光(guang)(guang)(guang)物質進(jin)行標記。免疫熒光(guang)(guang)(guang)技(ji)術(shu)(shu)可以(yi)用于研(yan)究細胞(bao)內蛋白質的定位(wei)和表達水平。

免(mian)疫(yi)熒光(guang)間接法測抗(kang)體(ti)實驗(yan)注意事項：1）熒光(guang)染(ran)色后一般在1h內(nei)完成(cheng)觀察(cha)，或于(yu)4℃保存4h，時間過長，會(hui)使(shi)熒光(guang)減(jian)弱(ruo)。2）每次試(shi)驗(yan)時，需(xu)設置(zhi)以(yi)下三(san)種對照：①陽(yang)性對照：陽(yang)性血(xue)清+熒光(guang)標(biao)記(ji)物②陰性對照：陰性血(xue)清+熒光(guang)標(biao)記(ji)物③熒光(guang)標(biao)記(ji)物對照：PBS+熒光(guang)標(biao)記(ji)物3.已知(zhi)抗(kang)原(yuan)標(biao)本片(pian)(pian)需(xu)在操作的(de)各個(ge)步(bu)驟中(zhong)，始(shi)(shi)終保持濕潤，避(bi)免(mian)干燥。4.所滴加的(de)待檢抗(kang)體(ti)標(biao)本或熒光(guang)標(biao)記(ji)物，應始(shi)(shi)終保持在已知(zhi)抗(kang)原(yuan)標(biao)本片(pian)(pian)上(shang)，避(bi)免(mian)因(yin)放置(zhi)不平使(shi)液體(ti)流失，從而造成(cheng)非特異性熒光(guang)染(ran)色。免(mian)疫(yi)熒光(guang)技術可以(yi)用于(yu)研究植物病(bing)理學和農業生產。COL-2免(mian)疫(yi)熒光(guang)染(ran)色

在實際工作中，熒(ying)光(guang)抗原技術(shu)應用較少，因此通常將其(qi)稱為熒(ying)光(guang)抗體技術(shu)或免疫(yi)(yi)熒(ying)光(guang)技術(shu)。collagenX(COL-10)免疫(yi)(yi)熒(ying)光(guang)試驗

細(xi)(xi)(xi)胞(bao)免疫熒光(guang)實(shi)驗注(zhu)意事項(xiang)：1、建議(yi)細(xi)(xi)(xi)胞(bao)直接(jie)種孔板，采用(yong)倒置(zhi)(zhi)熒光(guang)顯微鏡拍照，這(zhe)樣可(ke)以避(bi)免然后(hou)(hou)挑片(pian)(pian)(pian)時造(zao)成劃片(pian)(pian)(pian)或細(xi)(xi)(xi)胞(bao)片(pian)(pian)(pian)破損以及(ji)然后(hou)(hou)封片(pian)(pian)(pian)可(ke)能造(zao)成滑片(pian)(pian)(pian)等(deng)結(jie)果；2、若(ruo)實(shi)驗室只(zhi)(zhi)有正置(zhi)(zhi)熒光(guang)顯微鏡，則需要將細(xi)(xi)(xi)胞(bao)植(zhi)于細(xi)(xi)(xi)胞(bao)爬片(pian)(pian)(pian)。建議(yi)直接(jie)購買處理過的細(xi)(xi)(xi)胞(bao)爬片(pian)(pian)(pian)；若(ruo)只(zhi)(zhi)有普通細(xi)(xi)(xi)胞(bao)爬片(pian)(pian)(pian)，可(ke)以先將爬片(pian)(pian)(pian)于濃(nong)酸(suan)（濃(nong)酸(suan)腐(fu)蝕性強，注(zhu)意操作）或 75% 乙醇浸泡(pao)過夜，若(ruo)用(yong)酸(suan)浸泡(pao)過夜，第(di)二天先用(yong)自(zi)來水小心沖洗(xi)掉爬片(pian)(pian)(pian)殘留的酸(suan)液(ye)，若(ruo)用(yong) 75% 乙醇浸泡(pao)，則不需要此步(bu)驟(zou)。然后(hou)(hou)，用(yong)洗(xi)潔精搓洗(xi)爬片(pian)(pian)(pian)，然后(hou)(hou)用(yong)去離(li)子水清洗(xi)爬片(pian)(pian)(pian)。置(zhi)(zhi)于烘(hong)箱(xiang) 80 ℃ 烘(hong)干，再將爬片(pian)(pian)(pian)置(zhi)(zhi)于超(chao)凈臺造(zao)紫外消毒后(hou)(hou)備用(yong)。collagenX(COL-10)免疫熒光(guang)試驗

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