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 .
