FACTOR VIII
HRP Procedure

Cut formalin fixed paraffin sections at 5m and place on APES treated slides (Superfrost + or Surgipath X-tra)..

Heat immobilize sections in a 80C for 20 min.

Deparaffinize in 3 changes of xylene for 5 min each.

Hydrate in series of graded ethanols starting with 2 changes of absolute, one 95%, and ending with one 70%, all for 5 min each.

Rinse slides in distilled water for 2 min.

Place slides in a coplin or vertical staining jar and digest in 0.4% pepsin (0.4g + 100ml H20 + 500l 2N HCL), incubate in a  37C water bath for 15 min.

Rinse slides in 3 changes of distilled water.

Block endogenous peroxidase activity using 3% H2O2 for 5 min.

Rinse slides in 3 changes of distilled water.

Rinse in 3 changes of PBS for 5 min each.

Apply Normal Donkey Serum 1:5 for 20 min at room temp.

Drain excess serum and apply primary antibody rabbit anti-Factor VIII {Dako} 1:800 for 30 min at room temp.

Rinse in 3 changes of PBS for 5 min each.

Apply biotinylated Donkey-Rabbit Fab’2 secondary antibody 1:1000 {Jackson Immuno}. Incubate for 30 min at room temp.

Rinse in 3 changes of PBS for 5 min each.

Apply conjugated HRP streptavidin (Dako P397) 1:1000,   incubate for 30 min.

Rinse slides in 3 changes of PBS for 5 min each.

Apply stable DAB {Research Genetics} and incubate for 6 min at RT.

Rinse slides in 2 changes of distilled water.

Counterstain with dilute Harris hematoxylin 1:10 for 2 min.

Rinse slides until clear in distilled water.

Immerse slides in Scotts water for 1 min.

Rinse slides in  distilled water.

Dehydrate,  clear and mount with permount.