ACUITYAdvanced Protocol
ACUITYAdvanced system is recommended for use on formalin fixed paraffin embedded sections.
Positively charged slides recommended to securely adhere tissue.
Paraffin embedded sections must be de-paraffinized with xylene and rehydrated with a graded series of ethanolbefore staining.
DO NOT let specimen or tissue dry from this point on.
Optimal working dilution and incubation times are to bedetermined by the investigator.
We recommend Peroxidase Block, Catalog# 927401 or 927402.
If supplied by user, prepare as per recommended protocol (supplied by user for 931101, 931201, 930501).
When using ACUITYAdvanced hydrogen peroxide, incubate slides in 3% hydrogen peroxide blocking reagent for 10 minutes (hydrogen peroxide is provided with 930901, 931001, 930601 and 930701).
Rinse with distilled water.
Please refer to your antibody datasheet for recommended protocols if required.
For HIER we recommend HIER, Catalog # 928501 (order separately).
HIER or enzyme for digestion to be supplied by user.
Wash with PBS 2 minutes, 3 times.
Apply 2 drops (100 μL or enough volume to cover tissue section) of ACUITYAdvanced Reagent 1.
Incubate in a humidity chamber for 10 minutes.
Drain or blot off solution.
Do not rinse!
Apply 2 drops (100 μL or enough volume to cover tissue section) of primary antibody.
Incubate in a humidity chamber for 30-60 minutes.
Rinse with PBS 2 minutes, 3 times.
Apply 2 drops (100 μL or enough volume to cover tissue section) of ACUITYAdvanced Reagent 2.
Incubate in a humidity chamber for 15-20 minutes.
Rinse with PBS 2 minutes, 3 times.
Apply 2 drops (100 μL or enough volume to cover tissue section) of ACUITYAdvanced Reagent 3.
Incubate in a humidity chamber for 15 minutes.
Rinse with PBS 2 minutes, 3 times If supplied by user; prepare as per recommended protocol.
When using ACUITYAdvanced Chromogens (provided with kits 930901, 931001, 930601 and 930701) please reference Chromogen Preparation Table.
Rinse with distilled or tap water (AEC is alcohol soluble; do not dehydrate).
Counterstain with desired counterstain.
Mount and coverslip.
AEC chromogen should be prepared 1 part AEC chromogen to 50 parts AEC Substrate Buffer.
DAB chromogen should be prepared 1 part DAB Chromogen to 25 parts DAB Substrate Buffer.
The following table provides some sample preparation examples.
