DAB
Protocol
-
Stop IN SITU reaction by washing
with PBS
2x5min.
2x30min.
-
Apply primary antibody diluted
in Blocking Buffer incubate
2 hours at room temp. or O/N at 4º C.
-
Apply Biotin-conjugated Secondary
antibody (1:500 dilution for Jackson immunochemical abs. Or 1:200 for Vector
Lab. antibodies
incubate1 hour and 30 minutes at room temperature
-
Make ABC
reagent (at the beginning of this wash cycle)
1) Take 5ml PBT
2) Add 1 drop of reagent A, mix well.
3) Add 1 drop of reagent B, mix again
4) Let the mix stay at RT for 30min.
-
Apply ABC reagent for 1 hour
-
Make DAB
Reagent:
to 10mL of water add one tablet of UREA Hydrogen and one tablet of DAB.
- Vortex
- Add 500µl of solution to each slide
-
Monitor the Rxn. Under a dissecting
scope.
-
Stop reaction w/ PBS before
background is overwhelming:
- Wash slides with PBS - 6x5min. or O/N
-
Dehydrate slides in 30, 50,
70, 95 and 100% ethanol (5min. each), clear in xylene (30min.)
PBT:
(Phosphate Buffer with Tween)
1 liter PBS
2mL Tween 20
Blocking
Buffer:
40 mL PBT
400µL HINGS (heat inactivated Goat serum)
400µL HINDS (heat inactivated Donkey serum)
400¨µL 10% Triton x 100
ABCReagent
Vector Laboratories, Vectastain
ABC Kit Elite Rb IgG
PK-6101
DAB
Reagent
Sigma, Sigma Fast™ 3.3 Diaminobezidine
Tablet Sets
D-4293