I am using this protocol to stain intraepidermal nerve fibers in epidermis of a paraffin embedded 3 mm skin sections
I first deparrafinized and hydrated tissues, applued citrate buffer ph 6 antigen retrival in water bath for 1 hour
Incubate with 10% normal goat serum for 1-4 hours, primary polyclonal rabbit antibody against pgp 9.5 (1:50) for 24 hours at 4 degrees, secondary biotinylated giat anti rabbit (1:100), h2o2 3% block for 10-15 min, streptavidin-hrp (1:50-100) and fresh DAB and counterstain in hematoxylline
What is wrong? Why keratinicytes falsely stain with DAB?
N. B I diluted the primary, secondary and streptavidin-hrp in 1x PBS with 0.5% Tween-20
An updated image from Leica ICC50
What is wrong? Primary antibody diluent? Or concentration? Or Incubation time?
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