3 In Situ Nick End-Labeling: Light Microscopical
25
(Tap off excess buffer, wipe around specimen and apply enough
TdT reaction solution -biotin to cover specimen in moist chamber at 37°C)
11. Rinse the samples 3 times with 50mM PH 7.5 Tris-HCI for 5
mins each.
12. Immerse the samples in 0.3% HzOz in methanol for 15 mins
at RT to inactivate the endogeneous peroxidase. (Immerse
the samples into the cup which contains 1ml 30%HzOz and
99ml methanol)
13. Wash three times with 0.075% (v/v) Brij35/PBS for 5 mins
each.
Note: In this protocol, HzOz and antibody are washed with
0.075% Brij35/PBS which increase the efficiency of washing as
compared to only PBS.
14. After a brief washing with PBS, incubate with normal goat
IgG (0.5 mg/ml in 5% BSA/PBS) at RT for 1hr to reduce the
nonspecific binding of antibody to the samples.(Tap off excess buffer, wipe around specimen and apply normal goat
IgG to cover specimen in moist chamber at RT)
15. Incubate with HRP-conjugated goat anti-biotin (1/100 in
1 %BSA/PBS) at RT for 2hrs. (Tap off excess normal gout
IgG solution, wipe around specimen and apply enough
HRP-conjugated goat anti-biotin to cover specimen in moist
chamber at RT).
16. Wash with 0.075% (v/v) Brij35/PBS 4 times, 10 mins each,
then immerse in PBS.
TdT reaction
solution -
biotin
17. Visualize with DAB-HzOz and enhance with CoClz and Ni- DAB reaction
S04(NH4hS04 at RT for 5mins. (Immerse the samples into solution
the cup containing (DAB) reaction solution at RT for 5mins).
18. Wash with running water for short time and finally distilled
water for 5 mins.
19. Dehydration and mounting.
25
(Tap off excess buffer, wipe around specimen and apply enough
TdT reaction solution -biotin to cover specimen in moist chamber at 37°C)
11. Rinse the samples 3 times with 50mM PH 7.5 Tris-HCI for 5
mins each.
12. Immerse the samples in 0.3% HzOz in methanol for 15 mins
at RT to inactivate the endogeneous peroxidase. (Immerse
the samples into the cup which contains 1ml 30%HzOz and
99ml methanol)
13. Wash three times with 0.075% (v/v) Brij35/PBS for 5 mins
each.
Note: In this protocol, HzOz and antibody are washed with
0.075% Brij35/PBS which increase the efficiency of washing as
compared to only PBS.
14. After a brief washing with PBS, incubate with normal goat
IgG (0.5 mg/ml in 5% BSA/PBS) at RT for 1hr to reduce the
nonspecific binding of antibody to the samples.(Tap off excess buffer, wipe around specimen and apply normal goat
IgG to cover specimen in moist chamber at RT)
15. Incubate with HRP-conjugated goat anti-biotin (1/100 in
1 %BSA/PBS) at RT for 2hrs. (Tap off excess normal gout
IgG solution, wipe around specimen and apply enough
HRP-conjugated goat anti-biotin to cover specimen in moist
chamber at RT).
16. Wash with 0.075% (v/v) Brij35/PBS 4 times, 10 mins each,
then immerse in PBS.
TdT reaction
solution -
biotin
17. Visualize with DAB-HzOz and enhance with CoClz and Ni- DAB reaction
S04(NH4hS04 at RT for 5mins. (Immerse the samples into solution
the cup containing (DAB) reaction solution at RT for 5mins).
18. Wash with running water for short time and finally distilled
water for 5 mins.
19. Dehydration and mounting.