-
Paraffin section or
frozen section to water and
rinse in PBS-Tween 20 for 2x2 min.
-
Antigen Retrieval: perform
antigen retrieval using
IHC-TekTM
Epitope Retrieval Solution (Cat# IW-1100)
or
IHC-TekTM
Proteinase K Solution (Cat# IW-1101)if necessary.
-
Serum Blocking: incubate sections
in normal serum – species same as secondary antibody. Note:
since this protocol uses avidin-biotin detection system,
avidin/biotin
block may be needed based on tissue type.
-
Primary Antibody: incubate sections
in primary antibody at appropriate dilution in
IHC-TekTM
Antibody Diluent (Cat# IW-1000 or IW-1001) for 1 hour at room temperature or overnight. Note:
No serum blocking is needed if antibody diluent is used.
-
Rinse in PBS-Tween 20 for 3x2 min.
- Peroxidase Blocking: incubate
sections in
peroxidase blocking solution for 10 minutes at room
temperature. Note:
For acetone fixed frozen sections, perform this
peroxidase blocking step using 0.3% H2O2 in methanol
prior to primary antibody
incubation to
avoid tissue destruction.
-
Rinse in PBS-Tween 20 for 3x2 min.
-
Secondary Antibody: incubate
sections in
Biotinylated secondary antibody in PBS for 30
minutes at room temperature.
-
Rinse in PBS-Tween 20 for 3x2 min.
-
Detection: incubate sections in
ABC-Peroxidase Solution for 30 minutes at room
temperature.
-
Rinse in PBS-Tween 20 for 3x2 min.
-
Chromagen/Substrate: incubate
sections in
peroxidase
substrate solution.
-
Rinse in PBS-Tween 20 for 3x2 min.
-
Counterstain with
counterstain solution if desired.
-
Rinse in running tap water for 2-5
minutes.
-
Dehydrate through 95% ethanol for 1
minute, 100% ethanol for 2x3min.
-
Clear in xylene for 2x5min.
-
Coverslip with
mounting medium.