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.
