科研相關(guān)軟件

IHC

processSample

PreparationTissue

Collection

(and

Perfusion);Tissue

FixationTissue

EmbeddingSectioning

and

MountingEpitope

(Antigen)

RecoveryQuenching/Blocking

EndogenousActivityBlocking

Nonspecific

SitesIHC

processSample

LabelingImmunodetectionCounterstainingSealing

the

Stained

SampleSample

VisualizationSample

Preparation1.

Tissue

CollectionRapidly

p and

to

prevent

hematologic(interferingantigen)

，the

tissue

is

perfused,

or

rinsed

of

blood.冷凍切片和石蠟切片：冷凍切片：優(yōu)點(diǎn)：很好地保存組織抗原，抗原丟失少，自發(fā)熒光低；缺點(diǎn)：形態(tài)結(jié)構(gòu)差，定位不很清晰，易受樣品內(nèi)不穩(wěn)定化合物影響；石蠟切片優(yōu)點(diǎn)：組織形態(tài)結(jié)構(gòu)好，定位清晰，較大的組織易于使用；缺點(diǎn)：包埋過程中容易破壞組織抗原，使抗原的免疫活性有所降低，自發(fā)熒光較冰凍切片高；2.

Tissue

FixationFixation

prevents

autolysis

and

necrosis

of

tissue，preservesantigenicity，enhances

the

refractive

index

of

tissue

constituents，increase

the of

cellular

elements

to

tissue

processing.Stabilize

cell

morphology

and

tissue

architectureDisable

proteolytic

enzymesStrengthen

samples

to

withstand

further

processing

and

stainingProtect

samples

against

microbial

contamination

and

positionFixative

selection

consideration：Type

of

fixative

(formaldehyde,

glutaraldehyde,

etc.)Rate

of

penetration

and

fixationFixative

concentrationpHTemperaturePost-fixation

treatmentChemical

fixativesFormaldehyde-based

solutionNeutral

buffered

formalin

and

Bouin’s（Bouin’s

solution

contains

picric

acid

and

issuitable

for

use

on

all

tissue

except

kidney)

.Mercuric

chloride-based

fixativesTo e

poor

cytological

preservation.Precipitating

fixativesInclude

ethanol,

methanol

and

acetone.

They

precipita arge

protein

molecules

and

aregood

forcytological

preservation.

They

are

not

good

for

electron

microscopy,

though,because

they

cause

tissue

shrinkage.

And

penetrating

ability

of

ethanol

is

poor.Dimethyl

suberimidate

(DMS)Include

retention

of

immunoreactivity

of

the

antigen

and

the

lack

of

aldehyde

groupsthat

require

blocking.Physical

fixationDrying(Blood

smears),

Frozen；3.

Tissue

EmbeddingFixed

tissue

samples

are

embedded

in

paraffin

to

maintain

thenatural

sh

and

architecture

of

the

sample

during

long-termstorage

and

sectioning

for

IHC.Samples

too

sensitive

for

either

chemical

fixation

or

the

solventsused

to

remove

the

paraffin

are

encased

in

cryogenicembeddingmedium

and

then

snap-frozen

in

liquid

nitrogen.4.

Sectioningand

MountingTo

increase

adhesion

of

section，it’s

need

to

treat

glass

slides

with3-aminopropyltriethoxysilane

(APTS)

or

poly-L-lysine,

which

bothleave

amino

groups

on

the

surface

of

the

glass

to

which

the

tissuedirectly

couples.Alternatively,

slides

may

be

coated

with

physical

adhesives,including

gelatin,

egg

albumin

or

Elmer's

glue5.

Epitope

(Antigen)

Recovery液固定過程中，組織中的抗原蛋白與產(chǎn)生交聯(lián)（乙醇和氯化系列的固定劑與蛋白質(zhì)形成凝結(jié)物），使得抗原的決定簇被封閉。因此要打開組織抗原蛋白與

的交聯(lián)，以提高組織抗原的檢出率。Heat

(heat-induced

epitope

retrieval;

HIER)Enzymatic

degradation

(proteolytic-inducedepitope

retrieval;

PIER).EnzymeApproximateactivation

temperatureIncubationtimeProteinase

K25-37

°C5

min.Trypsin37

°C10min.Pepsin37

°C5–20

min.Protease

XXIV37

°C5–10

min.Pronase25-37

°C30

min.Proteolytic

enzymes

and

typical

incubation

conditions.組織中的粒細(xì)胞、單核細(xì)胞及紅細(xì)胞等存在內(nèi)源性過氧化物酶消除，可用3%過氧化氫作用10分鐘以消除；生物素和lectin可通過阻斷劑封閉；加入適當(dāng)?shù)姆忾]液封閉非特異性位點(diǎn)以及固定劑中的會(huì)和一抗反應(yīng)的aldehyde基團(tuán)；6.

Quenching/Blocking

EndogenousActivityFor

staining

approaches

that

depend

on

biotin,

peroxidases

or

phosphatases

fordetection

of ,

quenching

or

masking

endogenous

forms

of

theseproteins

prevents

false

positive

and

high

background

detection.Biotin，avidin

or

lectinEndogenous

biotin----liver,

mammary

gland,

adipose

tissue

and

kidney；Endogenous

avidin----cryostatsections；Endogenous

lectin------tissue；Use

blocker(biotin，avidin)

or

0.2

M

alpha-methyl

mannoside

or

Streptavidin(a)

Before (b)

Afterplex

(ABC)

binding

to

mast

cells

in

submucosa

(a)

before,

and

(b)

after

blockingfor

endogenous

avidin

binding

activity

(EABA).Peroxidases

and

phosphatases(a)

Before

(b)

AfterRed

blood

cells

showing

endogenous

peroxidase

activity

(a)

before,

and

(b)after

blocking

withthree

percent

hydrogen

peroxide.(a)

Before (b)

AfterPlacenta

showing

endogenous

alkaline

phosphatase

activity

(a)

before,and

(b)

after

blocking

with

levamisole.E

n

z

y

m

e:

Perox

id

a

s

eE

n

z

y

m

e:

Alk

al

in

e

P

h

os

p

h

at

a

s

e

*Red

Blood

CellsPlacentaIntestine

—

situated

between

cellularcomponents

of

mucosaGranulocytesProximal

tubules

of

kidneyEosinophilsOsteoblast

in

boneHepatocytesArterial

&

capillary

endothelial

cell

surfacesMuscleStromal

reticulum

cellsKidneyNeutrophilsMonocytesFollicle

and

mantle

zones

inmost

lymphoid

tissue*

Alkaline

Phosphatase

is

destroyed

by

routine

f

ixation

and

paraffin-

embeddingproceduresDual

endogenous

enzyme

blockHorseradish

peroxidase

and

alkalinephosphatase

labelsHydrogenperoxideHorseradish

peroxidase

labelLevamisole

+

chromogen

exceptintestinal

alkaline

phosphataseAlkaline

phosphatase

labelWeak

acid

(0.3N

HCl),including

intestinal

alkalinephosphataseAlkaline

phosphatase

labelEndogenous

enzymes

found

in

a

variety

of

cells

and

tissue

types.Commonendogenous

enzyme

blocking

reagents

for

horseradish

peroxidase

and

alkaline

phosphatase

systems.Sample

Labeling1.ImmunodetectionReporter

Selection：The

type

of

experimentThe

level

of

antigen

expressionWhether

ornot

signal tation

is

requiredThekinds

of

imaging

needed

(light

vs.epifluorescent

vs.

confocal)Thecost

of

reagents

and

equipmentChromogenic and

Fluorescent

reportersChromogenicreportersAntibody-HRP

conjugates

are

superior

to

antibody-AP

conjugates

with

respect

tothe

specific

activities

of

both

the

enzyme

and

antibody.Fluorescent

reportersmultiplex

stainingwith

high-resolution

fluorescent

microscopy（eg：confocal）Direct

vs.

Indirect

StainingIndirect

IHC

staining

amplifies

thesignal

over

direct

methods.2.Counterstaining免疫組織化學(xué)染色后需要復(fù)染細(xì)胞核，以將組織細(xì)胞結(jié)構(gòu)顯示出來，使免疫組織化學(xué)染色結(jié)果定位清晰。IHC結(jié)果根據(jù)顯色劑的不同而不同，有棕色、藍(lán)色和紅色，復(fù)染細(xì)胞核的顏色也因染液不同而不同，一般有藍(lán)色（蘇木素）、綠色（甲基綠）和紅色（核固紅）三種。應(yīng)根據(jù)顏色對(duì)比清晰的原則進(jìn)行搭配，常用的是DAB顯色呈棕色—Mayer`s蘇木素復(fù)染細(xì)胞核呈藍(lán)色。3.

Sealing

the

StainedSampleSealing

the

sample

by

mounting

a

coverslip

with

an

appropriatemountant

stabilizes

the

tissue

sample

and

stain.

An

antifade

reagent

should

also

be

included

if

fluorescent

detection

will

be

performed

toprolong

fluorescence

excitation.Sample

Visualization陽性結(jié)果應(yīng)定位在細(xì)胞中相應(yīng)的部位，在細(xì)胞膜表達(dá)的抗原陽性結(jié)果應(yīng)定位在細(xì)胞膜上，在其他部位的陽性反應(yīng)均為非特異性染色。不當(dāng)?shù)目乖迯?fù)會(huì)導(dǎo)致抗原在組織細(xì)胞中定位的改變。根據(jù)所檢測(cè)抗原的不同，抗原分別定位在：a）細(xì)胞膜，如LCA和UCHL1等；b）細(xì)胞質(zhì)，如Keratin

和Lysozyme等；c）細(xì)胞核，如PCNA和ER等。組織的周邊、刀痕、皺折等部位往往呈陽性表達(dá)，但絕大多數(shù)都是非特異性染色。染色結(jié)果呈

并非都是抗原不表達(dá)，要考慮是否與組織中的抗原受到破壞有關(guān)。組織切片背景深與下列因素有關(guān)：第一抗體濃度太高或孵育時(shí)間過長(zhǎng)，溫度過高。顯色劑DAB濃度過高或H2O2太多。正常

封閉之后、滴加第一抗體之前用了PBS洗?？贵w純度不高、抗體孵育切片后洗不干凈?？贵w的保存和稀釋抗體應(yīng)于低溫保存，不宜反復(fù)存放于4度和-20度之間。檢測(cè)試劑盒一般存放于4度，不宜于-20度保存，如長(zhǎng)時(shí)間不用可存放于-20度，解凍使用后則不能再存放于0度以下，反復(fù)凍融使得與抗體結(jié)合的酶容易分解，導(dǎo)致檢測(cè)的敏感度降低；濃縮的抗體在染色前應(yīng)根據(jù)說明書要求或優(yōu)化得出最佳工作濃度以進(jìn)行稀釋。日常工作量不多時(shí)，可將抗體稀釋至1:5~20，染色前再稀釋成工作液。濃縮液抗體保存的時(shí)間較長(zhǎng)，反之稀釋后的抗體保存的期限較短，如使用即用型抗體，經(jīng)過一定時(shí)間后應(yīng)注意其效價(jià)是否有所降低，避免出現(xiàn)假 染色；抗體的稀釋度抗體稀釋度最佳的抗體稀釋度可提高染色質(zhì)量，與周圍組織或細(xì)胞形成良好的染色對(duì)比。如果抗體濃度過高，反而減少抗體與抗原的結(jié)合，甚至導(dǎo)致假 結(jié)果，故各

每當(dāng)使用一種新抗體時(shí)，必須試用多種稀釋度，從中確定最佳工作濃度，以防假 結(jié)果的出現(xiàn)。由于標(biāo)本的固定、切片種類、稀釋液種類和濕盒等具體條件均可影響稀釋度，故在測(cè)試之前必須使這些條件穩(wěn)定。稀釋度應(yīng)選擇陽性反應(yīng)最強(qiáng)、背景

最弱的滴度?？贵w稀釋液SignalStain?

Antibody

Diluent#8112TBST/5%

normal

goat

serum

(#5425):

To

5

ml

1XTBST

add

250

μl

normal

goat

serum.PBST/5%

normal

goat

serum

(#5425):

To

5

ml1XPBST

add

250

μl

normal

goat

serum.1X

PBS/0.1%

Tween-20

(1X

PBST):

To

prepare

1

L

add100

ml

10X

PBS

to

900

ml

dH20.

Add

1

ml

Tween-20and

mix.10X

Phosphate

Buffered

Saline

(PBS):

To

prepare

1

Ladd

80

g

sodium

chloride

(NaCl),

2

g

potassium

chloride(KCl),

14.4

g

sodium

phosphate,

dibasic

(Na2HPO4)

and2.4

g

potassium

phosphate,

monobasic

(KH2PO4)

to

1

LdH2O.

Adjust

pH

to

7.4.滴加抗體注意事項(xiàng)切片在

封閉后，傾去多余

，便可滴加第一抗體。滴加前取出玻片，用吸水紙拭去組織周圍的液體，組織與細(xì)胞表面的液體要用濾紙條輕輕吸掉，但注意勿使組織干燥，盡快滴加抗體工作液1滴(約50ul)于組織表面。由于切片周圍無液體，滴加的抗體成為一個(gè)液滴，這樣既可節(jié)省抗體，又可增加抗原與抗體結(jié)合的機(jī)會(huì)。如果玻片上是培養(yǎng)細(xì)胞，應(yīng)在鏡下選擇一個(gè)理想的區(qū)域用免疫組織化學(xué)油筆在此區(qū)域

畫一圓圈，再滴加抗體，爾后應(yīng)輕輕搖動(dòng)切片數(shù)秒，以促進(jìn)抗體與組織待測(cè)抗原的結(jié)合，避免假

小區(qū)的出現(xiàn)。這一操作過程雖然簡(jiǎn)單，卻十分重要。加抗體時(shí)切忌加樣器或吸頭碰到組織或細(xì)胞，以免造成破損。酶標(biāo)抗體系統(tǒng)中的酶與顯色劑與抗體連接的酶主要有辣根過氧化物酶和堿性磷酸酶(HRP或AP),顯色劑有DAB、AEC、固紅和固藍(lán)等。HRP-----DAB和AEC等作顯色劑，DAB顯色呈棕色，AEC呈紅色；AP-------固紅和固藍(lán)作顯色劑，固紅顯色陽性物呈紅色，而固藍(lán)呈藍(lán)色。DAB顯色形成的沉淀物最穩(wěn)定和不易褪色，較為常用，因其是

物，故要避免接觸皮膚和污染環(huán)境。Available

Detection

Systems:

SignalStain?

Boost

IHC

Detection

Reagent

(HRP,

Rabbit)

#8114

SignalStain?

Boost

IHC

Detection

Reagent

(HRP,

Mouse)

#8125實(shí)驗(yàn)對(duì)照為證實(shí)抗體和檢測(cè)試劑盒效價(jià)是否可靠，染色操作是否正確，一般需要進(jìn)行實(shí)驗(yàn)對(duì)照，以避免試劑失效或操作失當(dāng)而出現(xiàn)假和假陽性，確保染色結(jié)果的可靠性。陽性對(duì)照:選用已知染色中度陽性以上的組織切片染色，陽性切片應(yīng)呈陽性，此外組織中的內(nèi)對(duì)照也是很好的陽性對(duì)照。初學(xué)者應(yīng)設(shè)陽性對(duì)照。2.對(duì)照:選用已知染色

的組織切片染色，其結(jié)果應(yīng)為

。一般，

對(duì)照和陽性對(duì)照同時(shí)進(jìn)行，或其中有陽性染色結(jié)果時(shí)才有意義。http://w

/technologies/ihc

controls.html染色操作注意事項(xiàng)第一抗體分為單克隆和多克隆抗體，檢測(cè)試劑盒中的第二抗體也有分為抗鼠和抗兔抗體，不能連接錯(cuò)，否則一抗、二抗連接不上而導(dǎo)致假的染色結(jié)果。抗體孵育時(shí)間隨孵育溫度升高而減少，但一般不超過37度、如果不是由于

急于發(fā)報(bào)告，一般一抗置于4度孵育過夜較佳。用于一種染色方法，因檢測(cè)試劑盒的不同其敏感性不同，第一抗體的稀釋度也不同。常見問題及解決非特異性背景染色常見原因操作過程中漂洗不充分加試劑后切片干燥組織切片折疊組織中所含過氧化物酶未阻斷組織中所含內(nèi)源性生物素未封閉組織抗原彌散切片黏附劑過厚蛋白封閉不充分內(nèi)源的lectin（ABC法中Avidin可與其結(jié)合）二抗的交叉或非特異反應(yīng)解決辦法每步步驟后沖洗3次，每次5分鐘防止切片干燥勿取折疊處觀察染色結(jié)果可再配新鮮3％H202，孵育時(shí)間延長(zhǎng)使用阻斷劑阻斷內(nèi)源性生物素組織及時(shí)固定，固定液要符合標(biāo)準(zhǔn)重新配制黏附劑涂片延長(zhǎng)封閉時(shí)間或再用2％BSA封閉0.2

M

alpha-methyl

mannoside封閉加入2%的與二抗同種屬常見問題及解決染色過強(qiáng)常見原因一抗?jié)舛冗^高或抗體孵育時(shí)間過長(zhǎng)孵育溫度過高，超過37℃二抗孵育時(shí)間過長(zhǎng)DAB顯色時(shí)間過長(zhǎng)或濃度過高解決辦法降低一抗?jié)舛然蚩s短一抗孵育時(shí)間一般室溫20