Vectashield mounting reagent

Cardiac stem cell populations were plated on 2-well chamber glass slides (10,000 cells/well) in their respective growth media (see Table 1) for a minimum of 24 hours. After incubation, slides were washed with PBS and fixed in 4% paraformaldehyde for 5 minutes at 4°C. Following fixation, the slides were washed twice with PBS and permeabolized in PBS plus 0.1% Triton X-100, 0.1 M Glycine for 3 minutes, then washed once with PBS and blocked with TNB (1X TN (Tris-HCl, NaCl) Buffer, 5 μg/mL blocking reagent (PerkinElmer, #FP1012)) for 30 minutes. Primary antibodies were diluted in TNB (see Online Table I) and incubated overnight at 4°C. The following day slides were washed twice with PBS. Fluorescently conjugated secondary antibodies were diluted in TNB (1:200) and incubated 1.5 hours at room temperature. For c-Kit staining a horseradish peroxidase (HRP)-linked secondary antibody (1:500) was used, followed by tyramide signal amplification (1:50) (PerkinElmer, #NEL753001KT). After washing twice with PBS, DAPI was included in a final wash to fluorescently label the nuclei, and slides were coverslipped with Vectashield® mounting reagent (Vector Laboratories, #H-1000). All slides were imaged using a Leica TCS SP8 confocal microscope. A table of antibodies and dilution ratios is available in Online Table I.

Cells were processed for confocal IF staining of EGFR as previously described (63 (link)). Briefly, cells were fixed in 4% methanol-free formaldehyde for 15 min at room temperature, and permeablized with 0.2% Triton-X-100. Cells were blocked in 5% normal goat serum diluted in 0.2% Tirton-X-100 for 1 hr at room temperature, and stained with primary antibody overnight at 4°C. Primary antibody: EGFR (SC-03, 1:100). Secondary antibody (Life technologies): Alexa Fluor 546 (confocal) and Alexa Fluor 488 (super resolution) for 30 min at room temperature. EGFR blocking peptides were incubated with primary antibody for 3 hours prior to use in this protocol (Santa Cruz). EGFR signal was pseudo-colored red in super resolution images. Cells were stained with DAPI for 5 min and then mounted in Vectashield mounting reagent (Vector Laboratories, Burlingame, CA). Confocal IF microscopy was performed using a Nikon A1RSi l microscope, and Z-slices were taken at 200 nm slices. Super resolution microscopy was performed using Nikon’s N-SIM (structured illumination microscope) at 100X (115 nm resolution).