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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