科研相关软件

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.冷冻切片和石蜡切片：冷冻切片：优点：很好地保存组织抗原，抗原丢失少，自发荧光低；缺点：形态结构差，定位不很清晰，易受样品内不稳定化合物影响；石蜡切片优点：组织形态结构好，定位清晰，较大的组织易于使用；缺点：包埋过程中容易破坏组织抗原，使抗原的免疫活性有所降低，自发荧光较冰冻切片高；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液固定过程中，组织中的抗原蛋白与产生交联（乙醇和氯化系列的固定剂与蛋白质形成凝结物），使得抗原的决定簇被封闭。因此要打开组织抗原蛋白与

的交联，以提高组织抗原的检出率。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.组织中的粒细胞、单核细胞及红细胞等存在内源性过氧化物酶消除，可用3%过氧化氢作用10分钟以消除；生物素和lectin可通过阻断剂封闭；加入适当的封闭液封闭非特异性位点以及固定剂中的会和一抗反应的aldehyde基团；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免疫组织化学染色后需要复染细胞核，以将组织细胞结构显示出来，使免疫组织化学染色结果定位清晰。IHC结果根据显色剂的不同而不同，有棕色、蓝色和红色，复染细胞核的颜色也因染液不同而不同，一般有蓝色（苏木素）、绿色（甲基绿）和红色（核固红）三种。应根据颜色对比清晰的原则进行搭配，常用的是DAB显色呈棕色—Mayer`s苏木素复染细胞核呈蓝色。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阳性结果应定位在细胞中相应的部位，在细胞膜表达的抗原阳性结果应定位在细胞膜上，在其他部位的阳性反应均为非特异性染色。不当的抗原修复会导致抗原在组织细胞中定位的改变。根据所检测抗原的不同，抗原分别定位在：a）细胞膜，如LCA和UCHL1等；b）细胞质，如Keratin

和Lysozyme等；c）细胞核，如PCNA和ER等。组织的周边、刀痕、皱折等部位往往呈阳性表达，但绝大多数都是非特异性染色。染色结果呈

并非都是抗原不表达，要考虑是否与组织中的抗原受到破坏有关。组织切片背景深与下列因素有关：第一抗体浓度太高或孵育时间过长，温度过高。显色剂DAB浓度过高或H2O2太多。正常

封闭之后、滴加第一抗体之前用了PBS洗。抗体纯度不高、抗体孵育切片后洗不干净。抗体的保存和稀释抗体应于低温保存，不宜反复存放于4度和-20度之间。检测试剂盒一般存放于4度，不宜于-20度保存，如长时间不用可存放于-20度，解冻使用后则不能再存放于0度以下，反复冻融使得与抗体结合的酶容易分解，导致检测的敏感度降低；浓缩的抗体在染色前应根据说明书要求或优化得出最佳工作浓度以进行稀释。日常工作量不多时，可将抗体稀释至1:5~20，染色前再稀释成工作液。浓缩液抗体保存的时间较长，反之稀释后的抗体保存的期限较短，如使用即用型抗体，经过一定时间后应注意其效价是否有所降低，避免出现假 染色；抗体的稀释度抗体稀释度最佳的抗体稀释度可提高染色质量，与周围组织或细胞形成良好的染色对比。如果抗体浓度过高，反而减少抗体与抗原的结合，甚至导致假 结果，故各

每当使用一种新抗体时，必须试用多种稀释度，从中确定最佳工作浓度，以防假 结果的出现。由于标本的固定、切片种类、稀释液种类和湿盒等具体条件均可影响稀释度，故在测试之前必须使这些条件稳定。稀释度应选择阳性反应最强、背景

最弱的滴度。抗体稀释液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.滴加抗体注意事项切片在

封闭后，倾去多余

，便可滴加第一抗体。滴加前取出玻片，用吸水纸拭去组织周围的液体，组织与细胞表面的液体要用滤纸条轻轻吸掉，但注意勿使组织干燥，尽快滴加抗体工作液1滴(约50ul)于组织表面。由于切片周围无液体，滴加的抗体成为一个液滴，这样既可节省抗体，又可增加抗原与抗体结合的机会。如果玻片上是培养细胞，应在镜下选择一个理想的区域用免疫组织化学油笔在此区域

画一圆圈，再滴加抗体，尔后应轻轻摇动切片数秒，以促进抗体与组织待测抗原的结合，避免假

小区的出现。这一操作过程虽然简单，却十分重要。加抗体时切忌加样器或吸头碰到组织或细胞，以免造成破损。酶标抗体系统中的酶与显色剂与抗体连接的酶主要有辣根过氧化物酶和碱性磷酸酶(HRP或AP),显色剂有DAB、AEC、固红和固蓝等。HRP-----DAB和AEC等作显色剂，DAB显色呈棕色，AEC呈红色；AP-------固红和固蓝作显色剂，固红显色阳性物呈红色，而固蓝呈蓝色。DAB显色形成的沉淀物最稳定和不易褪色，较为常用，因其是

物，故要避免接触皮肤和污染环境。Available

Detection

Systems:

SignalStain®

Boost

IHC

Detection

Reagent

(HRP,

Rabbit)

#8114

SignalStain®

Boost

IHC

Detection

Reagent

(HRP,

Mouse)

#8125实验对照为证实抗体和检测试剂盒效价是否可靠，染色操作是否正确，一般需要进行实验对照，以避免试剂失效或操作失当而出现假和假阳性，确保染色结果的可靠性。阳性对照:选用已知染色中度阳性以上的组织切片染色，阳性切片应呈阳性，此外组织中的内对照也是很好的阳性对照。初学者应设阳性对照。2.对照:选用已知染色

的组织切片染色，其结果应为

。一般，

对照和阳性对照同时进行，或其中有阳性染色结果时才有意义。http://w

/technologies/ihc

controls.html染色操作注意事项第一抗体分为单克隆和多克隆抗体，检测试剂盒中的第二抗体也有分为抗鼠和抗兔抗体，不能连接错，否则一抗、二抗连接不上而导致假的染色结果。抗体孵育时间随孵育温度升高而减少，但一般不超过37度、如果不是由于

急于发报告，一般一抗置于4度孵育过夜较佳。用于一种染色方法，因检测试剂盒的不同其敏感性不同，第一抗体的稀释度也不同。常见问题及解决非特异性背景染色常见原因操作过程中漂洗不充分加试剂后切片干燥组织切片折叠组织中所含过氧化物酶未阻断组织中所含内源性生物素未封闭组织抗原弥散切片黏附剂过厚蛋白封闭不充分内源的lectin（ABC法中Avidin可与其结合）二抗的交叉或非特异反应解决办法每步步骤后冲洗3次，每次5分钟防止切片干燥勿取折叠处观察染色结果可再配新鲜3％H202，孵育时间延长使用阻断剂阻断内源性生物素组织及时固定，固定液要符合标准重新配制黏附剂涂片延长封闭时间或再用2％BSA封闭0.2

M

alpha-methyl

mannoside封闭加入2%的与二抗同种属常见问题及解决染色过强常见原因一抗浓度过高或抗体孵育时间过长孵育温度过高，超过37℃二抗孵育时间过长DAB显色时间过长或浓度过高解决办法降低一抗浓度或缩短一抗孵育时间一般室温20