DNA DNAlacZ lacI O lacY

lacA

C E P

rep

T

CAP

repressor CAP site pol-binding site operator structural genes terminator

lac operonlac operon

lactose

cAMP

8

DNA DNAlacZ lacI O lacY

lacA

C E P

rep

galactose

indoxyl

T

CAP

repressor CAP site pol-binding site operator structural genes terminator

IPTG

X-Gal

- X-Gal chromogenic substrate- X-Gal chromogenic substratelacZ genelacZ gene 9

DNS DNAlacZ lacI O lacY

lacA

C E P

trans-acetylase

permease

rep

-gal

T CAP

pol

repressor CAP site pol-binding site operator structural genes terminator

lacZ genelacZ gene - X-Gal chromogenic substrate- X-Gal chromogenic substrate9

DNA DNA

lacZ lacI O lacY

lacA

C E P T

lacZ gene in cloninglacZ gene in cloning

lacZ -fragment

-fragment

lacZ- lacZ-

Foreign DNA

repressor CAP site pol-binding site operator structural genes terminator

10

7. Immunohistochemistry7. Immunohistochemistry

YY

YY Y

Y

nucleus nucleus

cytoplasm

Cell surface antigenes

anti-C antibody

Fluorescent dye

Rabbit anti-C Primary antibody

Goat anti-rabbitSecondary antibody

Direct method Indirect method

B

A

A

BB B

BB

A

A

AA

C C

CC

- immunofluorescence- immunofluorescence

Citoplasmic antigenes

cytoplasm

D

E

Cytoplasmic antigenes

D

E

Cell surface antigens

27

peroxidase

YYY

Y

nucleus

cytoplasm

Cell surfice antigens

peroxidase Rabbit anti-C primary antibody

Goat anti-rabbitsecondary antibody

Indirect methods

B

BB

A

A

A

C

C

Yperoxidase

peroxidase

peroxidase

Y

avidin

- peroxidase

- biotin – avidin – peroxidase

Anti-rabbit antibody (secondary antibody)

- immunoperoxidase-based methods- immunoperoxidase-based methods

DAB + H2O2 Ni-DAB + H2O2

cytoplazmic antigens

anti-rabbit antibody(secondary antibody)

D

E

Yanti-C antibody

Y

anti-C antibody

7. Immunohistochemistry7. Immunohistochemistry 28

biotin

biotin

avidin

ABC method (ABC = avidin-biotin complex)

anti-rabbit antibody(secondary antibody)

Y

peroxidase

Y

primary antibody

antigen

7. Immunohistochemistry7. Immunohistochemistry- immunoperoxidase-based methods- immunoperoxidase-based methods

29

DAB + H2O2 Ni-DAB + H2O2

YY

YYY

Ynucleus

cytoplasm

Alkaline phosphatase Rabbit anti-C primary antibody

Goat anti-rabbit secondary antibody

Indirect method

B

BB

A

A

A

C

C

- alkaline phosphatase

- digoxigenin – anti-digoxigenin – alkaline phosphatase

alk. phosph.

Y

Sheep anti-rabbit antibody

alk. phosphYY

DIG

Anti-DIG antibody

anti-C antibody

Cell surfice antigenscytoplasmic antigens

D

E

Y

Rabbit anti-C antibody

30

YYalk. phosph

DIG

Anti-DIG antibody

anti-C antitbody

Y

anti-rabbit antibody

7. Immunohistochemistry7. Immunohistochemistry