A CAR RNA FISH assay reveals functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue
Eichholz Karsten, Li Alvason Zhenhua, Tareen Semih U.
“PrePrint: Immunohistochemistry To combine IHC with the CAR RNA FISH procedure we followed a previously described protocol with minor modification( ).. After the last RNA target was developed (step Amp4 in the RNAscope Multiplex Fluorescent Assay version 1, TSA amplification step in the RNAscope Multiplex Fluorescent Assay version 2), the cells and tissue sections were washed with PBS and blocked with 5% donkey or goat serum (Jackson ImmunoResearch), 2% BSA (Gemini Bio-Products) +0.2% Triton X-100 (SigmaAldrich) in 5X Casein (Vector Laboratories) for 30 min, the mouse anti-huCD19 antibody (dilution 1:100, Clone ID: UMAB103, origene) overnight at 4°C for the mouse xenograft tissues.. Slides were washed, incubated with secondary donkey anti-mouse IgG-Alexa 647 or Alexa 488 (1:100 Molecular Probes/ThermoFisher Scientific) or a goat anti-mouse BV480.. “