ab14174 (1/1000) staining SPARC in human substanta nigra using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of the cytoplasm and extracellular matrix.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH 6.0 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.

Immunohistochemical analysis of E14 and E16 mouse choroid plexus tissue, staining SPARC with ab14174. Sections were blocked with blocking agents and incubated with primary antibody (1/10000) overnight at 4°C. Staining was detected using DAB.