Immunohistochemistry Protocol for Beta Amyloid Products using USA Detection Kit
Clear Slides .
Removes paraffin and hydrates the tissue .
Note : If using frozen sections , allow slides to come to room temperature for 15 minutes & proceed to step ( F ) only
A . Xylene 5 minutes in each of ( 3 ) different 250mL containers
B . 100 % alcohol 5 minutes in each of ( 3 ) different 250mL containers
C . 95 % alcohol 3 minutes in ( 1 ) 250mL container
D . 70 % alcohol 3 minutes in ( 1 ) 250mL container
E . water 1 minutes in each of ( 3 ) different 250mL containers
F . H2O2 ( 3 % ) 15 minutes in ( 1 ) 250mL container .
Rinse slides with lab grade water .
Note : Lab grade filtered water such as injection grade , cell culture grade , Reverse Osmosis De - Ionization ( RODI ) .
Antigen Retrieval ( refer to product datasheet , not always required ) 70 % Formic Acid – incubate the slides for 20 minutes at room temperature .
Note : This antigen retrieval step is harsh on the tissue .
If using frozen sections reduce time to 5 - 10 minutes or omit if tissue falls off the slide .
Rinse Slides with 1X PBS .
Apply serum block for at least 5 minutes .
Do NOT wash after this step .
Blot off serum block .
Apply primary antibody – dilute to 1 mg / mL in PBS .
Incubate primary antibody 60 minutes at room temperature .
Rinse slides with 1X PBS .
Apply USA Linking reagent - 20 minutes incubation .
Rinse slides with 1X PBS .
Apply Labeling Reagent – 20 minutes incubation .
Rinse with 1X PBS .
Apply chromogen – 5 minutes incubation .
Dilute according to manufacturer’s instructions
AEC Chromogen : 20mL AEC chromogen + 1mL AEC substrate buffer .
DAB Chromogen : 40 mL DAB chromogen + 1mL DAB substrate buffer .
Rinse slides with lab grade water .
Submerge slides in Mayer’s Hematoxylin for 30 seconds .
Rinse under running lab grade water for 1 minute or until water is clear .
Submerge slides in Bluing Reagent for 1 minute .
Rinse under running lab grade water for 1 minute .
Clear slides . Dehydrate the tissue .
95 % alcohol 3 minutes in ( 1 ) 250mL container 100 % alcohol 5 minutes in each of ( 3 ) different 250mL container .
Xylene 5 minutes in each of ( 3 ) different 250mL container .
Cover slip slide using Permanent Aqueous Mounting Medium ( SIG - 31010 ) .
Note : Do not use xylene based mount with AEC Chromogen as it will dissolve the chromogen
