Dissections of pupal stages until P4 (for pupal staging, see Bainbridge and Bownes [1981] (link)) were performed after prefixation with 3.7% formaldehyde for 30 min with microsurgery scissors in toto. To dissect pharate adult stages, the pupal cases were removed before prefixation. For antibody stainings, the dissected animals were fixed in 3.7% formaldehyde for 10 min, washed three times for 30 min in PBT (PBS + 0.1% Tween 20), blocked in 10% BSA (Serva, 11930.04) for 1 h, and incubated with the primary antibodies, goat polyclonal anti-GFP (GeneTex, GTX26673, 1:1,000) and rabbit polyclonal anti-RFP (Rockland, 600-401-379S, 1:500) for 2 d at 4°C. To visualize filamentous actin, Atto 647N–conjugated phalloidin (1:1,000, Sigma-Aldrich, 65906) was added to the antibody solution. After incubation with the primary antibodies, the dissections were washed three times for 30 min in PBT and incubated with the respective secondary antibodies, Alexa Fluor 488–conjugated donkey anti-goat (Abcam, ab150129) and Alexa Fluor 555–conjugated donkey anti-rabbit (Abcam, ab150074), in 1:200 dilution overnight at 4°C. Finally, the stained animals were washed three times for 30 min in PBT and embedded into Vectashield containing DAPI (Vectorlabs, H-1200).
Atto 647n conjugated phalloidin
Manufactured by Merck Group
Atto 647N–conjugated phalloidin is a fluorescent dye-labeled chemical compound used for the detection and visualization of actin filaments in biological samples. It binds specifically to F-actin, allowing the identification and localization of the cytoskeletal structures in cells.
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2 protocols using atto 647n conjugated phalloidin
1
Pupal Stage Dissection and Antibody Staining
2
Imaging Myo7a in Mouse Inner Ears
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Dissected inner ears from Myo7afl/fl and Myo7afl/flMyo15-cre+/- mice (n = 5 and n = 8 mice, respectively) were perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 45 min at 4 °C. Cochleas were microdissected, rinsed three times for 10 min, and incubated for 1 h at room temperature in PBS supplemented with 20% normal horse serum and 0.3% Triton X-100. The samples were then incubated overnight with the primary antibody: rabbit anti-myosin7a68 (link) (1/500) in PBS supplemented with 1% horse serum. The samples were rinsed three times for 10 min in PBS, and then incubated for 1 h with ATTO-488-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich, #18772, 1:500 dilution). Actin was labeled with ATTO-647N–conjugated phalloidin (Sigma-Aldrich, #65906, 1:200 dilution). Samples were then mounted in Fluorsave (Calbiochem, USA). The z-stack images were captured with a Zeiss LSM-700 confocal microscope equipped with a Plan Apochromat 63X/NA 1.4 oil immersion lens (Carl Zeiss, Jena, Germany).
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