Alexafluor 488 tyramide

Adult C57BL/6J or Myd88^−/− mice were injected intramuscularly (i.m.) with 50 μl PBS or AAVrh32.33 single-stranded viruses (1 × 10¹⁰ or 1 × 10¹¹ vg per animal) encoding GFP or hFIX in the left quadriceps muscle. 21 d later, the animals were sacrificed and a portion of the quadriceps was fixed in 10% formalin overnight and transferred to 70% ethanol. For histology, muscle samples were processed at Dana-Farber/Harvard Cancer Center Specialized Histopathology Services and Beth Israel Deaconess Medical Center histology core facilities, embedded in paraffin, and stained for CD8 (1:500, D4W2Z, CST) and granzyme B (1:500, polyclonal, catalog # AF1865, R&D). Antibodies were tagged with AlexaFluor 488 Tyramide or AlexaFluor 647 Tyramide (B40957 and B40958, Thermo Fisher). All immunohistochemistry was performed on the Leica Bond automated staining platform using the Leica Biosystems Refine Detection Kit with citrate antigen retrieval. Muscle sections also were stained for GFP (1:800, polyclonal, catalog # ab6556, Abcam) using the Leica Biosystems Refine Detection Kit (DAB chromogen) with EDTA antigen retrieval. For rh32.33.FIX injected mice, plasma samples were obtained 14 d, 28 d, 42 d and 60 d after 1 × 10¹¹ vg AAV administration and hFIX expression was measured by ELISA as previously described.