All tissues were fixed overnight at room temperature in 10% neutral buffered
formalin, transferred to 70% EtOH, processed, and paraffin embedded. For
immunohistochemical staining, tissue was sectioned at 5μM, deparaffinized and
rehydrated prior to antigen retrieval in boiling citrate-based buffer (0.01 mol/L citric
acid, pH 6.8). Endogenous peroxidases were quenched with 3% H2O2,
followed by blocking in 5% goat serum and incubation with primary antibodies (seeSupplementary Material for details).
Bound antibodies were detected with the Vector M.O.M. peroxidase or fluorescein
Immunodetection Kits (Vector Laboratories, CA), using SigmaFast diaminobenzidene as a
peroxidase substrate or AlexaFluor594 (Jackson ImmunoResearch, PA) for immunofluorescence.
Nuclei were counterstained with hematoxylin followed by Permount (Fisher Scientific)
mounting or immunofluorescence visualized with ProLong Gold Antifade Reagent with DAPI
(Invitrogen, OR).
formalin, transferred to 70% EtOH, processed, and paraffin embedded. For
immunohistochemical staining, tissue was sectioned at 5μM, deparaffinized and
rehydrated prior to antigen retrieval in boiling citrate-based buffer (0.01 mol/L citric
acid, pH 6.8). Endogenous peroxidases were quenched with 3% H2O2,
followed by blocking in 5% goat serum and incubation with primary antibodies (see
Bound antibodies were detected with the Vector M.O.M. peroxidase or fluorescein
Immunodetection Kits (Vector Laboratories, CA), using SigmaFast diaminobenzidene as a
peroxidase substrate or AlexaFluor594 (Jackson ImmunoResearch, PA) for immunofluorescence.
Nuclei were counterstained with hematoxylin followed by Permount (Fisher Scientific)
mounting or immunofluorescence visualized with ProLong Gold Antifade Reagent with DAPI
(Invitrogen, OR).