Immunohistochemistry Protocol for Ultra Streptavidin Detection Kits (USA)
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% alcohol 5 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 using # 1 Blocking Reagent (included in 50 test kit only), or 927401/927402 (sold separately) for 150 and 500 test kit.
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). 
Retrieve-ALL (927901, 928201, 927601) or Sodium Citrate (928501, 928601). 
Heat slides in microwave on high for 1 minute 40 seconds in the appropriate retrieval solution at 1X. 
Reduce to low power and simmer 10 minutes in the microwave.
Allow to cool on the bench top for 10 minutes.
Rinse with lab grade water.
Rinse Slides with 1X PBS (926201). 
Note: Other antigen retrievals could include EDTA, Proteinase K, Pepsin, protease VIII – follow antibody manufacturer’s instructions.
Apply #2 Blocking Reagent (blue in color) for at least 5 minutes at room temperature.
Do NOT wash after this step.
Blot off serum block 6 mL predilute antibodies are ready to use, do not dilute 1 mL concentrate products can be diluted >1:40 in PBS or other antibody diluent. 
If using a non-BioLegend antibody, dilute according to the manufacturer’s instructions.
Incubate primary antibody 20-60 minutes at room temperature (refer to incubation time listed on the datasheet).
Rinse slides with 1X PBS.
Apply #4 Linking Reagent (yellow in color) – and incubate slides for 20 minutes at room temperature.
Rinse slides with 1X PBS.
Apply #5 Labeling Reagent (orange in color) – and incubate slides for 20 minutes at room temperature Rinse with 1X PBS.
Apply chromogen and incubate slides for 5 minutes at room temperature.
A. AEC Chromogen: 20μL AEC chromogen + 1mL AEC substrate buffer (1:50 Dilution) 
B. DAB Chromogen: 40 μL DAB chromogen + 1mL DAB substrate buffer (1:25 Dilution) 
Note: Not all USA Kits contain chromogen.
If using a non-BioLegend chromogen, dilute and incubate according to the manufacturer’s instructions.
Rinse slides with lab grade water.
Submerge slides in Hematoxylin for 30 seconds (not provided).
Rinse under running lab grade water for 1 minute or until water is clear.
Submerge slides in Bluing Reagent for 1 minute (not provided).
Rinse under running lab grade water for 1 minute.
Clear slides: Dehydrate the tissue.
A. 95% alcohol 3 minutes in (1) 250mL container 
B. 100% alcohol 5 minutes in each of (3) different 250mL container 
C. Xylene 5 minutes in each of (3) different 250mL container.
Cover slip slide using Permanent Aqueous Mounting Medium or Xylene Based medium.
Note: Do not use xylene based mount with AEC Chromogen as it will dissolve the chromogen.
