細(xì)胞成像的教案

細(xì)胞成像的教案第1頁(yè)/共73頁(yè)細(xì)胞成像講座內(nèi)容1. 細(xì)胞成像的原理和應(yīng)用2. 細(xì)胞成像的實(shí)驗(yàn)流程3. 細(xì)胞成像的實(shí)驗(yàn)設(shè)計(jì)4. 細(xì)胞成像的常見問題解析第2頁(yè)/共73頁(yè)通過巧妙結(jié)合免疫反應(yīng)的特異性，實(shí)現(xiàn)在組織、細(xì)胞、亞細(xì)胞原位水平檢測(cè)各種抗原物質(zhì)。樣本抗原一抗二抗標(biāo)記物細(xì)胞成像的原理第3頁(yè)/共73頁(yè)ICC/IF 細(xì)胞免疫熒光?

使用抗體對(duì)細(xì)胞中目標(biāo)抗原進(jìn)行定位、定性或定量分析Live

cell

imaging 活細(xì)胞成像?

通過連續(xù)采集顯微鏡下的圖像來研究活細(xì)胞的結(jié)構(gòu)與功能熒光顯微成像第4頁(yè)/共73頁(yè)細(xì)胞成像的發(fā)展熒光素FITC/TRITC/PECy2/Cy3/Cy5AlexaFluor?

/DyLight?QuantumDotAlexa

Fluor?

is

aregisteredtrademark

of

Molecular

Probes,

Inc,

a

Thermo

FisherScientificCompany.成像技術(shù)熒光顯微鏡共聚焦顯微鏡超分辨率熒光顯微技術(shù)第5頁(yè)/共73頁(yè)細(xì)胞成像的應(yīng)用Immunocytochemistry/

immunofluorescence細(xì)胞免疫熒光的應(yīng)用?

觀察目標(biāo)蛋白表達(dá)豐度?

觀察亞細(xì)胞定位?

觀察蛋白的共定位?

很好的藥物篩選模型定性、定量定位定位定量第6頁(yè)/共73頁(yè)Oncotarget,

Vol.

7,

No.

51.

細(xì)胞成像的應(yīng)用：觀察目標(biāo)蛋白表達(dá)豐度DMSO DNPDAPI CLU DAPI CLUMMP-9mergemergeMMP-9第7頁(yè)/共73頁(yè)2.

細(xì)胞成像的應(yīng)用：觀察目標(biāo)蛋白的亞細(xì)胞定位Stem

Cells.

2012

October;30(10):

2065–2075.

doi:10.1002/stem.1139.第8頁(yè)/共73頁(yè)3.

細(xì)胞成像的應(yīng)用：觀察細(xì)胞顯微結(jié)構(gòu)Ke

Xu,

Guisheng

Zhong

et

at.,

2013STOM超分辨顯微鏡第9頁(yè)/共73頁(yè)4.

細(xì)胞成像的應(yīng)用：觀察蛋白的共定位Journalofvirology,

July

1998,

p.5717-5727第10頁(yè)/共73頁(yè)5.

細(xì)胞成像的應(yīng)用：藥物篩選模型Xu

et

al,

Nature,

2008Ascreening

approach.

Screens

can

be

carried

outincells

byexamining

multipleprotein

markers

and

functionalchanges

(for

example,cellmorphology)

using

automated

high-content

imaging.第11頁(yè)/共73頁(yè)細(xì)胞成像講座內(nèi)容1. 細(xì)胞成像的發(fā)展和原理2. 細(xì)胞成像的實(shí)驗(yàn)流程3. 細(xì)胞成像的實(shí)驗(yàn)設(shè)計(jì)4. 細(xì)胞成像的常見問題解析第12頁(yè)/共73頁(yè)細(xì)胞成像的實(shí)驗(yàn)流程固定fixation通透permeabilization封閉blocking一抗孵育Primary

Ab

incubation二抗孵育Secondary

Ab

incubation復(fù)染和封片counterstaining/

mounting第13頁(yè)/共73頁(yè)細(xì)胞成像的實(shí)驗(yàn)流程樣本固定固定fixation通透permeabilization封閉blocking一抗孵育Primary

Abincubation二抗孵育Secondary

Abincubation復(fù)染和封片counterstaining/mounting固定：有效維持細(xì)胞的形態(tài)，結(jié)構(gòu)與組分。使細(xì)胞被“保存”和穩(wěn)定在接近生理培養(yǎng)的狀態(tài)。Livecell

imaging 活細(xì)胞成像不要固定細(xì)胞第14頁(yè)/共73頁(yè)細(xì)胞成像的實(shí)驗(yàn)流程樣本固定交聯(lián)試劑甲醛溶液多聚甲醛戊二醛有機(jī)溶劑甲醇乙醇丙酮? 交聯(lián)試劑有利于保護(hù)細(xì)胞結(jié)構(gòu)，但是會(huì)產(chǎn)生蛋白交聯(lián)，可能會(huì)降低抗原性? 預(yù)冷的有機(jī)溶劑固定會(huì)吸去脂類使細(xì)胞脫水、蛋白變性，這種固定方法不需要額外的通透步驟第15頁(yè)/共73頁(yè)細(xì)胞成像的實(shí)驗(yàn)流程樣本固定固定方式的不同:

case1.

細(xì)胞膜定位100%Methanol4%PFAITAMDAPITubulinMerge第16頁(yè)/共73頁(yè)細(xì)胞成像的實(shí)驗(yàn)流程樣本固定固定方式的不同:

case2.

細(xì)胞膜定位100%Methanol4%PFAEGFR

(phospho

Y1068)DAPITubulinMerge例：Anti-EGFR

(phospho

Y1068)

antibody

[Y38]

(ab32430)第17頁(yè)/共73頁(yè)細(xì)胞成像的實(shí)驗(yàn)流程樣本通透固定fixation通透permeabilization封閉blocking一抗孵育Primary

Abincubation二抗孵育Secondary

Abincubation復(fù)染和封片counterstaining/mounting通透化1. 當(dāng)抗體檢測(cè)細(xì)胞內(nèi)蛋白時(shí)（包括抗原表位在胞內(nèi)段的跨膜蛋白），需要對(duì)細(xì)胞進(jìn)行通透。2. 將樣品在含0.1-0.2%

Triton

X-100的PBS

中孵育10

分鐘。3. 使用預(yù)冷的丙酮固定則不需要通透。第18頁(yè)/共73頁(yè)細(xì)胞成像的實(shí)驗(yàn)流程樣本封閉? 封閉非特異性結(jié)合可用10%

血清，5%

BSA,

或

Protein

Block

(ab156024)；使用Biotin標(biāo)記物,需要封閉內(nèi)源Avidin、Biotin,

可用Endogenous

Avidin/BiotinBlocking

Kit

(ab64212)；? 使用熒光標(biāo)記物,

當(dāng)使用甲醛固定時(shí)，用含0.3M

甘氨酸的封閉液封閉,

也可以選用相應(yīng)的熒光蛋白封閉液,

如FITC

ProteinBlocking

Agent

(PBA)

(ab128980)固定fixation通透permeabilization封閉blocking一抗孵育Primary

Abincubation二抗孵育Secondary

Abincubation復(fù)染和封片counterstaining/mounting第19頁(yè)/共73頁(yè)? 濃度梯度確定抗體的最佳工作濃度，使特異性染色最大化并減少背景? 避免抗體反復(fù)凍融，降低抗體效價(jià)? 室溫孵育2小時(shí)或4℃孵育過夜? 使用1%BSA

inPBS

作為抗體稀釋液建議濃度細(xì)胞成像的實(shí)驗(yàn)流程固定fixation通透permeabilization封閉blocking一抗孵育Primary

Abincubation二抗孵育Secondary

Abincubation復(fù)染和封片counterstaining/mounting一抗孵育第20頁(yè)/共73頁(yè)固定fixation通透permeabilization封閉blocking一抗孵育Primary

Abincubation二抗孵育Secondary

Abincubation復(fù)染和封片counterstaining/mounting?

二抗一般帶有熒光素，注意避光保存?

確定合適的濃度，避免無信號(hào)或背景過高?

一般室溫孵育半小時(shí)以上?

多蛋白檢測(cè)時(shí)，選擇偶聯(lián)不同熒光素的二抗，避免串色細(xì)胞成像的實(shí)驗(yàn)流程二抗孵育第21頁(yè)/共73頁(yè)ab150061 DonkeyAnti

-

Rabbit IgG

H&L(Alexa

Fluor?488)preadsorbed一抗的種屬Rabbit一抗的亞型IgG

H&L實(shí)驗(yàn)應(yīng)用Alexa

Fluor?

488二抗選擇一抗的種屬來源：rabbit;

mouse;

chicken;

rat

等一抗的抗體亞型：IgG;

IgM;

IgA;

IgY，IgE是否需要預(yù)吸附PreadsorbedMinimal

cross-reactivity:

Human,

Mouse,

RatAlexa

Fluor?

is

a

registered

trademark

of

Molecular

Probes,

Inc,

a

Thermo

Fisher

Scientific

Company.細(xì)胞成像的實(shí)驗(yàn)流程第22頁(yè)/共73頁(yè)預(yù)吸附（Pre-adsorbed）的二抗排除二抗與不同種屬的一抗結(jié)合，減少非特異性結(jié)合，降低背景。Preadsorbed

Alexa

Fluor?405

conjugated

secondaryantibodiesPreadsorbedAlexa

Fluor?

488

conjugated

secondaryantibodiesPreadsorbed

Alexa

Fluor?555

conjugated

secondaryantibodiesPreadsorbed

Alexa

Fluor?594

conjugated

secondaryantibodiesPreadsorbed

Alexa

Fluor?647

conjugated

secondaryantibodiesPre-adsorbed

HRP

conjugated

secondaryantibodiesPre-adsorbed

biotinylatedsecondaryantibodies第23頁(yè)/共73頁(yè)23

November

2018復(fù)染和封片固定fixation通透permeabilization封閉blocking一抗孵育Primary

Abincubation二抗孵育Secondary

Abincubation復(fù)染和封片counterstaining/mounting封片劑? 維持樣品狀態(tài)，防止樣品變干? 防熒光淬滅高折射率（RefractiveIndex），提高圖像的分辨率；細(xì)胞成像的實(shí)驗(yàn)流程含DAPI的防熒光淬滅封片劑

ab104139第24頁(yè)/共73頁(yè)抗體質(zhì)量問題第25頁(yè)/共73頁(yè)抗體質(zhì)控的金標(biāo)準(zhǔn)特異性敲除驗(yàn)證靈敏度RabMAb?抗體一致性重組技術(shù)第26頁(yè)/共73頁(yè)抗體的金標(biāo)準(zhǔn)特異性敲除驗(yàn)證：

~

1500個(gè)產(chǎn)品結(jié)果CRISPR/Cas9

genome-editingKi67

knockout

HAP1

cellsWild-type

HAP1

cellsAnti-Ki67

antibody

[EPR3610]

(ab92742)Anti-alpha

Tubulin抗體[DM1A]-

Microtubule

Marker(Alexa

Fluor?

594)

(ab195889)第27頁(yè)/共73頁(yè)靈敏性HighLowAffinityLowHighSpecificityRabbit

PolyRabMAbMouse

mAbRabMAb：~13000個(gè)產(chǎn)品結(jié)果抗體的金標(biāo)準(zhǔn)Kd

distributor第28頁(yè)/共73頁(yè)抗體的金標(biāo)準(zhǔn)靈敏性CDX2MousemonoclonalRabbitmonoclonalE-CadherinHer2第29頁(yè)/共73頁(yè)抗體的金標(biāo)準(zhǔn)一致性重組抗體：

~

13000個(gè)產(chǎn)品結(jié)果23

November

2018重組抗體? 穩(wěn)定持續(xù)供貨? 批間差異小? 生產(chǎn)周期短第30頁(yè)/共73頁(yè)Copyright

?

2013

Abcam

plc.IHC

batch

testing

of

human

lungNSCLC

stained

with

anti-PD-L1(ab205921)

at

2

μg/mL.A

=

control,

B

–F

=

differentbatches.

Allbatches

showedconsistent

results.抗體的金標(biāo)準(zhǔn)一致性第31頁(yè)/共73頁(yè)蛋白數(shù)據(jù)庫(kù)

www.uni這個(gè)數(shù)據(jù)庫(kù)是關(guān)于該蛋白的詳細(xì)信息介紹，比如翻譯后修飾、亞細(xì)胞定位、組織特異性等，也可以找到Atlas數(shù)據(jù)庫(kù)鏈接名稱，基因名，別名，剪切片段名亞細(xì)胞定位翻譯后修飾組織表達(dá)特異性同源異構(gòu)體的氨基酸序列第32頁(yè)/共73頁(yè)Atlas數(shù)據(jù)庫(kù)鏈接第33頁(yè)/共73頁(yè)Atlas數(shù)據(jù)庫(kù)鏈接第34頁(yè)/共73頁(yè)細(xì)胞成像講座內(nèi)容1. 細(xì)胞成像的發(fā)展和原理2. 細(xì)胞成像的實(shí)驗(yàn)流程3. 細(xì)胞成像的實(shí)驗(yàn)設(shè)計(jì)4. 細(xì)胞成像的常見問題解析第35頁(yè)/共73頁(yè)細(xì)胞成像的實(shí)驗(yàn)設(shè)計(jì)Case

1.選擇熒光Case

2.雙染或多染Case

3.直標(biāo)一抗Case

4.細(xì)胞器定位第36頁(yè)/共73頁(yè)1212Case

1.

選擇熒光? 激發(fā)光、發(fā)射光? 波譜特征：波峰、波寬? 熒光強(qiáng)度? 穩(wěn)定性? 兼容性：溶液、pH第37頁(yè)/共73頁(yè)?光穩(wěn)定性高?明亮的顏色?獨(dú)立的波段?光譜覆蓋廣?良好水溶性?對(duì)pH不敏感Alexa

Fluor?

is

a

registered

trademark

of

Molecular

Probes,

Inc,

a

Thermo

Fisher

Scientific

Company.Case

1.

選擇熒光Alexa

Fluor 488495熒光染料Ex(nm)Em(nm)顏色FITC495519GreenCy2?489506Green?519GreenDyLight?

488

493518Green第38頁(yè)/共73頁(yè)用兩種/多種不同熒光染料標(biāo)記的抗體同時(shí)檢測(cè)兩種/多種抗原Case

2.

雙染或多染/science/connectome-project/brainbow第39頁(yè)/共73頁(yè)? 避免抗體間交叉反應(yīng)? 避免熒光素之間的串色：

AlexaFluor

?

488/

568/

594/

647

nmCase

2.

雙染或多染第40頁(yè)/共73頁(yè)NeuNRabbitmonoclonal

[EPR12763]

to

NeuN

(ab177487)GoatAnti-Rabbit

IgG

Fc

(Alexa

Fluor?

488)

preadsorbed

(ab150097)GFAPChicken

polyclonal

to

GFAP

(ab4674)GoatAnti-Chicken

IgY

H&L(Alexa

Fluor?594)

preadsorbed(ab150176)Mouse

cerebellumNeuN在成熟神經(jīng)元中特異表達(dá)GFAP在膠質(zhì)細(xì)胞中特異表達(dá)Alexa

Fluor?

is

a

registered

trademark

of

Molecular

Probes,

Inc,

a

Thermo

Fisher

Scientific

Company.Case

2.

雙染或多染第41頁(yè)/共73頁(yè)Case

3.

直標(biāo)一抗直標(biāo)一抗：一抗上直接偶聯(lián)熒光素。?排除物種間交叉反應(yīng)，有效降低非特異性結(jié)合?操作簡(jiǎn)單，快速?避免二抗選擇的復(fù)雜性?有相應(yīng)的熒光素偶聯(lián)試劑盒配套，快速高效?適用于多重染色?但是，沒有信號(hào)放大效果第42頁(yè)/共73頁(yè)雙標(biāo)：兩個(gè)直標(biāo)一抗alpha

smooth

muscle

Actin-Alexa

Fluor?

488

(ab197240)alpha

Tubulin-Alexa

Fluor?

594

(ab195889)DAPIHeLa

cells

were

fixed

with

4%

formaldehyde

(10

min),

andpermeabilized

in

0.1%

Triton

X-100

for

5minutes.Alexa

Fluor?

is

a

registered

trademark

of

Molecular

Probes,

Inc,

a

Thermo

Fisher

Scientific

Company.Case

3.

直標(biāo)一抗第43頁(yè)/共73頁(yè)Alexa

Fluor?

is

a

registered

trademark

of

Molecular

Probes,

Inc,

a

Thermo

Fisher

Scientific

Company.Case

3.

直標(biāo)一抗雙標(biāo)：直標(biāo)一抗+非直標(biāo)一抗Vimentin

-

Alexa

Fluor?

647(ab195878)betaTubulin

(ab6046)Goat

Anti-Rabbit

IgG

H&L

(AlexaFluor?

488)

preadsorbed

(ab150081)DAPIHeLa

cells,

fixed

with

4%

formaldehyde

(10

min),permeabilized

in

0.1%

Triton

X-100

for

5

minutes.23

November

2018第44頁(yè)/共73頁(yè)Case

3.

直標(biāo)一抗偶聯(lián)試劑盒?

快捷、方便地將標(biāo)記物偶聯(lián)到抗體：一步標(biāo)記方法，不需要分步操作。?

可標(biāo)記少量抗體：低至10μ/kits/antibody-conjugation-kitsConjugateEx/Em

(nm)ColorProduct

code?Dylight

405400/420ab201798?Dylight

350353/432ab201797AMCA350/445ab195224?Dylight

488493/518ab201799FITC494/520ab188285?Cy3550/570ab188287?Dylight

550562/576ab201800Rhodamine561/588ab188286?Texas

Red595/615ab195225?Dylight

594593/618ab201801?Dylight

633638/658ab201802?Cy5647/665ab188288?Dylight

650652/672ab201803?Dylight

680682/715ab201804?Dylight

755754/776ab201805?Dylight

800770/794ab201806第45頁(yè)/共73頁(yè)使用細(xì)胞器marker來進(jìn)行定位Case

4.

細(xì)胞器定位Mouse

monoclonal

[AF18]

to

Calnexin-ERMembrane

Marker

(Alexa

Fluor?647)(ab202572)Mouse

monoclonal

[DM1A]

to

alpha

Tubulin-

Microtubule

Marker(Alexa

Fluor?

488)(ab195887)Mouse

monoclonal

[9B6]

to

Giantin-

Golgi

Marker(ab37266)Rabbitpolyclonal

to

betaTubulin-

Loading

Control

(ab6046)第46頁(yè)/共73頁(yè)23

November

2018Case

4.

細(xì)胞器定位高爾基體Golgi

reassembly

stackingprotein

2內(nèi)質(zhì)網(wǎng)Protein

disulfide

isomerasefamily

A線粒體Anti-TUFM線粒體Citrate

synthase過氧化物酶體ATP-binding

cassette,sub-family

D

(ALD),

member

3內(nèi)涵體Vacuolar

protein

sorting26

homolog

A細(xì)胞質(zhì)膜Anti-Ezrin焦點(diǎn)黏連Anti-ZyxinHuman

Protein

Atlas第47頁(yè)/共73頁(yè)NuclearERGolgiCase

4.

細(xì)胞器定位使用細(xì)胞器特異的染料來進(jìn)行定位CytoPainter

Golgi/ER

Staining

Kit

(ab139485)liverat

astrocytecellsER

(red),

Golgi

(green),

Nuclear(blue).第48頁(yè)/共73頁(yè)Case

4.

細(xì)胞器定位使用細(xì)胞器特異的染料來進(jìn)行定位：CytoPainter61+

targets

to

multipleorganelles

and

structures? 操作便捷? 光穩(wěn)定強(qiáng)? 無種屬限制? 避免非特異結(jié)合? 基本都適用于活細(xì)胞標(biāo)記CellmembraneCytoplasmNucleusPlasma

membraneCytoskeletonNuclearIntercellular

junctionCentrosomeCentromereCaveolaeEndosomeNuclear

poreERNuclear

envelopeGolgiNucleolarMitochondriaHeterochromatinLysosomePeroxisomeRibosome第49頁(yè)/共73頁(yè)細(xì)胞成像講座內(nèi)容1. 細(xì)胞成像的發(fā)展和原理2. 細(xì)胞成像的實(shí)驗(yàn)流程3. 細(xì)胞成像的實(shí)驗(yàn)設(shè)計(jì)4. 細(xì)胞成像的常見問題解析第50頁(yè)/共73頁(yè)細(xì)胞成像的結(jié)果分析理想的結(jié)果? 樣本組織結(jié)構(gòu)完整? 熒光信號(hào)強(qiáng)、穩(wěn)定? 無非特異性染色? 低背景第51頁(yè)/共73頁(yè)細(xì)胞成像的結(jié)果分析常見問題? 沒有信號(hào)? 高背景染色? 不正確的信號(hào)第52頁(yè)/共73頁(yè)沒有信號(hào)改善微弱或沒有染色信號(hào)? 沒有足夠的一抗與目標(biāo)蛋白結(jié)合延長(zhǎng)孵育時(shí)間、增加抗體濃度? 固定步驟遮蔽了抗體識(shí)別表位（使用福爾馬林和多聚甲醛固定劑）縮短固定時(shí)間，更換固定劑? 蛋白位于細(xì)胞質(zhì)/核內(nèi)，抗體不能穿透質(zhì)/核膜在封閉液和抗體稀釋液中加入通透劑? 靶細(xì)胞中沒有目標(biāo)蛋白建議做陽(yáng)性對(duì)照第53頁(yè)/共73頁(yè)例2.

Rabbitmonoclonal

[EP854(2)Y]

to

Histone

H2A.X(phospho

S139)

-

ChIPGrade(ab81299)HeLa

cell

linewithout

treatmentHeLa

cell

linetreated

with

H2O2

(100

μM)

for

2h沒有信號(hào)Case1.

確認(rèn)實(shí)驗(yàn)?zāi)Ｐ?，是否需要?duì)細(xì)胞進(jìn)行處理第54頁(yè)/共73頁(yè)4%

PFAfixedKi67TubulinMergeCase2.

確認(rèn)細(xì)胞狀態(tài)DAPINew

A431

cell

lineOld

A431

cell

line沒有信號(hào)第55頁(yè)/共73頁(yè)改善高背景染色結(jié)果? 沒有封閉非特異性結(jié)合或封閉不充分延長(zhǎng)封閉時(shí)間，更換換封閉劑（使用與二抗宿主來源的血清）? 一抗?jié)舛冗^高建立濃度梯度預(yù)實(shí)驗(yàn)，在單染的體系里面找到最佳抗體濃度? 二抗發(fā)生了非特異性結(jié)合使用陰性對(duì)照（不加一抗，只加二抗），使用預(yù)吸附的二抗? 經(jīng)醛類固定劑處理的細(xì)胞會(huì)產(chǎn)生自由醛基封閉液中加入0.3M

甘氨酸? 洗滌不充分在洗滌緩沖液中加入

0.2%Tween

20? 細(xì)胞培養(yǎng)過于密集優(yōu)化細(xì)胞生長(zhǎng)條件高背景染色第56頁(yè)/共73頁(yè)Confocal數(shù)據(jù)采集過程影響因素

曝光時(shí)間長(zhǎng)

激光強(qiáng)度高Pinhole太大非Sequential模式Alexa

Fluor?

is

a

registered

trademark

of

Molecular

Probes,

Inc,

a

Thermo

Fisher

Scientific

Company.SequentialPictures

were

obtained

from

.au/myscope/confocal/virtual/confocal/multichannelimaging.phpSimultaneousHoechst33342Osteoclast高背景染色第57頁(yè)/共73頁(yè)案例分析綠色：Pin1

(ab224527)紅色：alpha

Tubulin(ab7291)藍(lán)色：DAPI

(ab104139)Pin1和Tubulin抗體質(zhì)量很好

綠色熒光蛋白信噪比低，可以增加激光強(qiáng)度，縮短曝光時(shí)間，微調(diào)cutoff

值和

LUTs

DAPI

過曝，可降低激光強(qiáng)度，縮短曝光時(shí)間第58頁(yè)/共73頁(yè)案例分析優(yōu)化前優(yōu)化后第59頁(yè)/共73頁(yè)確認(rèn)相關(guān)信息? 細(xì)胞培養(yǎng)條件是否有利于目標(biāo)蛋白表達(dá)與正確定位（e.g.

celljunction

protein）? 文獻(xiàn)檢索目標(biāo)蛋白在特定細(xì)胞中的亞細(xì)胞定位? 嘗試改變固定條件，進(jìn)一步確認(rèn)通透化處理是否恰當(dāng)? 染色期間務(wù)必要避免細(xì)胞干燥，因?yàn)檫@可能引起假陽(yáng)性? 如果有條件，使用

KO

或

RNAi樣本染色信號(hào)不正確第60頁(yè)/共73頁(yè)固定方法的影響Rabbitmonoclonal

[EPR1755(2)]

to

APG5L/ATG5

(ab108327)

胞漿定位是正確的染色信號(hào)不正確第61頁(yè)/共73頁(yè)Rabbitmonoclonal

[SP6]

to

Ki67

(ab16667)Wild-type

HAP1

cellsKi67

knockout

HAP1

cells染色信號(hào)不正確確認(rèn)抗體的特異性：敲除（KO）驗(yàn)證第62頁(yè)/共73頁(yè)Rabbitmonoclonal

[EP1510Y]

to

RNA

polymeraseII

CTD

repeatYSPTSPS

POLR2A

(phosphoS1801)(ab76292)HeLa

cell

lineHeLa

cell

line

treated

with

AP染色信號(hào)不正確確認(rèn)抗體的特異性：用堿性磷酸酶（AP）鑒定磷酸化信號(hào)的特異性第63頁(yè)/共73頁(yè)小 結(jié)細(xì)胞成

像常見

問題沒信號(hào)、弱信號(hào)高背景信號(hào)不正確樣本材料操作第64頁(yè)/共73頁(yè)Qdot量子點(diǎn)技術(shù)的優(yōu)點(diǎn)：*

多種顏色：顏色取決于量子點(diǎn)的大小，在同一激發(fā)波長(zhǎng)下，可發(fā)出多種激發(fā)光，達(dá)到同時(shí)檢測(cè)多種指標(biāo)的要求*

熒光時(shí)間長(zhǎng)：熒光時(shí)間較普通熒光分子延長(zhǎng)數(shù)千倍，便于長(zhǎng)期追蹤和保存結(jié)果*

檢測(cè)方便：最簡(jiǎn)單的熒光顯微鏡即可進(jìn)行檢測(cè)*

安全：細(xì)胞毒性低、可用于活細(xì)胞或者體內(nèi)研究*

應(yīng)用范圍廣：可用于多領(lǐng)域和多儀器量子點(diǎn)技術(shù)的用途：1、觀測(cè)活細(xì)胞里多個(gè)蛋白質(zhì)的活動(dòng)：量子點(diǎn)

(quantumdots)

現(xiàn)已可取代傳統(tǒng)染色法，成為細(xì)胞內(nèi)的熒光標(biāo)記物，可進(jìn)行長(zhǎng)時(shí)間、多分子同時(shí)檢測(cè)。參考文獻(xiàn)：Jaiswal

JKet

al.

Nature

Biotechnology

2002

Dec22、熒光標(biāo)記：

這種新型熒光材料的出現(xiàn)為眾多生物問題的研究帶來的曙光。量子點(diǎn)可與多種分子進(jìn)行偶聯(lián)，如Protein

A、抗體、鏈霉親和素等，作為熒光探針，檢測(cè)特定靶分子的分布和功能。第65頁(yè)/共73頁(yè)QdotSeminal

developments

in

the

story

of

nanocrystal

technology

emerged

in

the

early

1980s

from

thelabs

of

Louis

Brus

at

Bell

Laboratories

and

of

Alexander

Efros

and

A.I.

Ekimov

of

the

Yoffe

Institute

in

St.Petersburg

(then

Leningrad)

in

the

former

Soviet

Union.

Dr.

Brus

and

his

collaborators

experimentedwith

nanocrystal

semiconductor

materials

and

observed

solutions

ofstrikingly

different

colors

madefrom

thesame

substance.

This

work

contributed

to

the

understanding

of

the

quantum

confinementeffect

that

explains

the

correlation

between

size

and

color

for

these

nanocrystals.

Two

scientists

fromBell

Labs—Dr.

Moungi

Bawendi

andDr.

Paul

Alivisatos—moved

to

MIT

and

UCBerkeley,

respectively,and

continued

investigating

quantum

dot

optical

properties.

These

researchers

found

ways

to

makethe

quantum

dots

water

soluble.

Theyalso

discovered

that

adding

apassivating

inorganic

"shell"around

the

nanocrystals,

and

then

shining

blue

light

on

them,

caused

the

quantum

dots

to

light

upbrightly.

Invitrogen

is

the

exclusive

licensee

of

several

of

their

discoveries.第66頁(yè)/共73頁(yè)QdotQdot

BioconjugatesQdot

bioconjugate

is

a

generic

termto

describe

Qdot

nanocrystals

coupled

to

proteins,oligonucleotides,

small

molecules,

etc.,which

are

used

to

direct

binding

of

the

quantum

dots

totargets

of

interest.

Examples

of

Qdot

bioconjugates

include

streptavidin,

protein

A,

and

biotinfamilies

of

conjugates.

Qdot

bioconjugates

are

oftenused

assimple

replacements

for

analogousconventional

dye

conjugates

when

their

unique

performance

characteristics

are

required

toachieve

optimal

results.Most

dye

conjugates

aresynthesized

byattaching

one

or

more

fluorophores

to

a

single

biomolecule;however,

thelarge

surface

areaafforded

by

the

nanocrystal

fluorophore

allows

simultaneousconjugation

of

many

biomolecules

to

a

single

Qdot

nanocrystal.

Advantages

conferred

by

thisapproach

include

increased

avidity

for

targets,the

potentialfor

cooperative

binding

in

some

cases,and

the

use

of

efficient

signal

amplification

methodologies.

For

example,

combining

biotin-functionalized

products

with

thestreptavidin

labels

allows

for

successive

enhancements

in

signal

via"sandwiching"

(streptavidin/biotin/streptavidin/etc.)

following

an

initial

labeling

step.第67頁(yè)/共73頁(yè)QdotQdot

Nanocrystal

StructureFundamentally,

Qdot

nanocrystals

are

fluorophores—substances

that

absorb

photons

of

light,

then

re-emitphotons

ata

differentwavelength.

However,

they

exhibit

some

important

differences

ascompared

totraditional

fluorophores

such

as

organic

fluorescent

dyes

and

naturally

fluorescent

proteins,

ends

there.Qdot

nanocrystals

are

nanometer-scale

(roughly

protein-sized)

atomclusters,

containing

from

afewhundred

to

a

few

thousand

atoms

of

asemiconductor

material(cadmium

mixed

with

selenium

ortellurium),whichhas

been

coated

with

an

additional

semiconductor

shell

(zinc

sulfide)

to

improve

theoptical

properties

of

the

material.

These

particles

fluoresce

in

a

completely

differentway

thandotraditional

fluorophores,

withoutthe

involvement

of

->*

electronic

transitions.At

the

heart

of

the

fluorescence

of

Qdot

nanocrystals

is

the

formation

of

excitons,

or

Coulomb

correlatedelectron-hole

pairs第68頁(yè)/共73頁(yè)QdotNanocrystal

emission

series

formulticolor

analysisWith

their

broad

excitation

and

narrow,

symmetricemission

properties

(Figure

5),

the

QdotRnanocrystals

require

only

a

single

excitation

source(typically<450

nm,

usually

405),

facilitatingmultiplex

analysis

of

multiple