Protocols
PDL-1 mAb IHC-P in mouse tissues
Immunohistochemistry (IHC-P) labelling of murine formalin fixed tissues using the rabbit PDL-1 monoclonal antibody.
3 μm tissue sections were prepared from formalin-fixed paraffin embedded tissues and mounted onto Superfrost®plus slides and dried overnight at 55oC. The sections were placed in an automated Autostainer Link 48 immunostaining platform and stained according to the following protocol.
After being deparaffinized, antigen retrieval was performed using the high pH Agilent buffer at 96 ºC during 20 minutes (Dako) and washed before blocking any endogenous peroxidase with 3% hydrogen peroxide. After washing, slides were then incubated with the rabbit anti-PD-L1 (EPR20529, 1/2000, Abcam, ab213480) during 40 minutes, after another wash the slides were incubated with the EnVision anti-Rabbit HRP visualization system. The immunohistochemical reaction was revealed using the 3, 30-diaminobenzidine tetrahydrochloride (DAB) solution included in the Envision FLEX kit and cell nuclei were counterstained with Mayer’s Hematoxylin included in the kit. The resulting slides were then dehydrated, washed and mounted with permanent mounting medium for microscopic evaluation.
Appendix
Autostainer Link 48 Dako
Rabbit monoclonal anti- PD-L1 (EPR20529, 1/2000, Abcam, ab213480)
EnVision™ FLEX, high pH (Dako)
EnVision™ anti rabbit K4003