ab12242(1:1000) staining Chromogranin B in human pancreas (IgG inset) using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of Chromogranin B in secretory compartments of the pancreas.
Insert panel depicts negative control (no primary antibody).
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX.
Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.