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. Xylene5 minutes in each of (3) different 250mL containers
B. 100% alcohol5 minutes in each of (3) different 250mL containers
C. 95% alcohol3 minutes in (1) 250mL container
D. 70% alcohol3 minutes in (1) 250mL container
E. water1 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
