Immunohistochemistry Protocol for Keratin Antibodies
Clear Slides : Removes paraffin and hydrates the tissue .
A . Xylene : 5 minutes in each of ( 3 ) different 250mL containers
B . 100 % alcohol 5 minutes in each of ( 3 ) different 250mL containers
C . 95 % alcohol 3 minutes in ( 1 ) 250mL container
D . 70 % alcohol 3 minutes in ( 1 ) 250mL container
E . Water 1 minutes in each of ( 3 ) different 250mL containers
F . H2O2 ( 3 % ) 15 minutes in ( 1 ) 250mL container .
Rinse slides with lab grade water .
Note : Lab grade filtered water such as injection grade , cell culture grade , Reverse Osmosis De - Ionisation ( RODI ) .
Heat slides in 1X Sodium Citrate solution for 1 minute 25 seconds on high power in microwave .
Reduce to low power and simmer for 10 minutes in microwave .
Remove from microwave and allow slides to cool on the bench top for 10 minutes .
Rinse slides with lab grade water .
Apply serum block for at least 5 minutes .
Do NOT wash after this step .
Blot off serum block .
Apply primary antibody ( see recommended dilution from datasheet ) .
Incubate primary antibody 60 minutes at room temperature .
Rinse slides with 1X PBS .
Apply USA Linking reagent - 20 minutes incubation .
Rinse slides with 1X PBS .
Apply Labeling Reagent – 20 minutes incubation .
Rinse with 1X PBS .
Apply chromogen – 5 minutes incubation .
Dilute according to manufacturer’s instructions .
AEC Chromogen : 20μL AEC chromogen + 1mL AEC substrate buffer .
Rinse slides with lab grade water .
Submerge slides in Mayer’s Hematoxylin for 30 seconds .
Rinse under running lab grade water for 1 minute or until water is clear .
Submerge slides in Bluing Reagent for 1 minute .
Rinse under running lab grade water for 1 minute .
Cover slip slide using Permanent Aqueous Mounting Medium ( SIG - 31010 ) .
Note : do not use xylene based mount with AEC Chromogen as it will dissolve the chromogen .
