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 ethanol before 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 .
