TITLE: ACUITYAdvanced Protocol
AUTHORS: Sam Li

[DESCRIPTION]
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[STEPS]
?. [Peptide Exchange:]
ACUITYAdvanced system is recommended for use on formalin fixed paraffin embedded sections.
?. [Peptide Exchange:]
Positively charged slides recommended to securely adhere tissue.
?. [Peptide Exchange:]
Paraffin embedded sections must be de-paraffinized with xylene and rehydrated with a graded series of ethanol before staining.
?. [Peptide Exchange:]
DO NOT let specimen or tissue dry from this point on. Optimal working dilution and incubation times are to be determined by the investigator.
?. [Staining Protocol: Peroxidase Blocking]
We recommend Peroxidase Block, Catalog#927401 or 927402. If supplied by user, prepare as per recommended protocol (supplied by user for 931101, and 931201).
?. [Staining Protocol: Peroxidase Blocking]
When using ACUITYAdvanced hydrogen peroxide, incubate slides in 3% hydrogen peroxide blocking reagent for 10 minutes (hydrogen peroxide is provided with 931001).
?. [Staining Protocol: Peroxidase Blocking]
Rinse with distilled water.
?. [Heat Induced Epitope Retrieval (HIER) or enzymatic digestion]
Please refer to your antibody datasheet for recommended protocols if required.
?. [Heat Induced Epitope Retrieval (HIER) or enzymatic digestion]
For HIER we recommend HIER, Catalog # 928502 (order separately). HIER or enzyme for digestion to be supplied by user.
?. [Heat Induced Epitope Retrieval (HIER) or enzymatic digestion]
Wash with PBS 2 minutes, 3 times.
?. [ACUITYAdvanced Reagent 1 (Serum Block)]
Apply 2 drops (100 µL or enough volume to cover tissue section) of ACUITYAdvanced Reagent
?. [ACUITYAdvanced Reagent 1 (Serum Block)]
Incubate in a humidity chamber for 10 minutes.
?. [ACUITYAdvanced Reagent 1 (Serum Block)]
Drain or blot off solution. Do not rinse.
?. [Primary Antibody (supplied by user)]
Apply 2 drops (100 µL or enough volume to cover tissue section) of primary antibody.
?. [Primary Antibody (supplied by user)]
Incubate in a humidity chamber for 30-60 minutes.
?. [Primary Antibody (supplied by user)]
Rinse with PBS 2 minutes, 3 times.
?. [ACUITYAdvanced Reagent 2 (Boost)]
Apply 2 drops (100 µL or enough volume to cover tissue section) of ACUITYAdvanced Reagent 2.
?. [ACUITYAdvanced Reagent 2 (Boost)]
Incubate in a humidity chamber for 15-20 minutes.
?. [ACUITYAdvanced Reagent 2 (Boost)]
Rinse with PBS 2 minutes, 3 times.
?. [ACUITYAdvanced Reagent 3 (HRP Polymer)]
Apply 2 drops (100 µL or enough volume to cover tissue section) of ACUITYAdvanced Reagent 3.
?. [ACUITYAdvanced Reagent 3 (HRP Polymer)]
Incubate in a humidity chamber for 15 minutes.
?. [ACUITYAdvanced Reagent 3 (HRP Polymer)]
Rinse with PBS 2 minutes, 3 times.
?. [Chromogen (supplied by user for 931101, 931201)]
If supplied by user; prepare as per recommended protocol.
?. [Chromogen (supplied by user for 931101, 931201)]
When using ACUITYAdvanced Chromogens (provided with kit 931001), please reference Chromogen Preparation Table.
?. [Chromogen (supplied by user for 931101, 931201)]
Rinse with distilled or tap water (AEC is alcohol soluble; do not dehydrate).
?. [Counterstain and mount (supplied by user)]
Counterstain with desired counterstain.
?. [Counterstain and mount (supplied by user)]
Mount and coverslip.
?. [Chromogen Preparation]
AEC chromogen should be prepared 1 part AEC chromogen to 50 parts AEC Substrate Buffer.
?. [Chromogen Preparation]
DAB chromogen should be prepared 1 part DAB Chromogen to 25 parts DAB Substrate Buffer. The following table provides some sample preparation examples. ACUITYAdvanced Chromogen Preparation Table  ABCD1ChromogenDesired Working VolumeChromogen VolumeBuffer Volume2AEC5 mL0.1 mL AEC5 mL310 mL0.2 mL AEC10 mL425 mL0.5 mL AEC25 mL56DAB10 mL0.4 mL DAB10 mL720 mL0.8 mL DAB20 mL830 mL1.20 mL DAB30 mLIntended Use: Research Use Only (RUO); This product is sold for laboratory research use only, not for human or in vivo use.Warranty/Conditions: BioLegend products may not be resold or modified for resale without prior written approval.Storage: Store between 2-8°C.
ABCD1ChromogenDesired Working VolumeChromogen VolumeBuffer Volume2AEC5 mL0.1 mL AEC5 mL310 mL0.2 mL AEC10 mL425 mL0.5 mL AEC25 mL56DAB10 mL0.4 mL DAB10 mL720 mL0.8 mL DAB20 mL830 mL1.20 mL DAB30 mL