As an example, staining using ABC system is described below. If recommended procedure after secondary antibody reaction is indicated by ABC kit supplier, please follow the recommended procedure.
1) Deparaffinize sections by xylene, and hydrate by using series of ethanol solutions.
2) Wash sections with DW for over 5 min.
3) Denature the endogenous peroxidase by using 0.3 to 1.0% H2O2 solution.
4) Rinse the section with DW for 5 min and incubate for 5 min in PBS two times.
5) Coat entire tissue sections with blocking solution, and incubate for 30 minutes at room temperature in a humid chamber. There is no limitation of the species of blocking agents.
6) Dilute primary antibody by one of three solutions of IMMUNO SHOT Immunostaining to the concentration recommended by supplier.
7) After removal of blocking solution from sections, apply diluted primary antibody solution, and incubate for 1 hours at RT or overnight at 4C in humid chamber. Incubation time varies from antibody to antibody.
8) Wash sections with PBS for 5 min three times.
9) Dilute biotinized secondary antibody by one of three solutions of IMMUNO SHOT Immunostaining according to dilution factor recommended by supplier.
10) Apply diluted biotinized secondary antibody solution, and incubate for 30 min in humid chamber. Then, wash sections with PBS for 5 min three times. (it is recommended to prepare avidin-biotin-peroxidase complex solution during this incubation time)
11) Apply the complex solution to the sections, and incubate for 30 min in humid chamber.
12) Wash sections with PBS for 5 min three times, and incubate with chromatic substrate solutions.
13) Terminate reaction and mount the section using mounting medium. Observe by microscope.
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.