i'm  trying to double stain tissue section with different host antibody by using this protocol.

Ø the tissue section were place in coplin jar contain ice cold acetone  for 1 hour.

Ø the slides were washed with 0.3% PBST buffer for 3 times with 5 min interval by  using  1 % BSA for blocking at 1 hour in a dark place .

Ø After 1 hour blocking the blocking solution were discard and again wash with 0.3% PBST solution.

Ø Incubate the tissue section in the mixture of two primary antibodies (Nestin mouse monoclonal antibody and Rabbit polyclonal to doublecortin ) in 1% BSA in PBST in a humidified chamber for overnight  at 4oC.

Ø next day the primary antibody was washed with 0.3% PBST solution and use Anti - mouse alaxa flour 488 and Anti - Rabbit Alaxa Flour 555 (1 µL/ 1 ml)  as a secondary antibody for 1 hour at room temperature.

Ø after 1 hour the secondary antibody was discard and again wash with 0.3% PBST solution then used DAPI  mounting solution and fix the slide and take image by using immunoflourescence microscope .

 but it not shows signals.

can any one please tell me why ?