Anti-Myc tag antibody [9E10] (ab32)

(A-D) Representative examples of body forming AtMORC7-MYC, AtMORC4-MYC, At-MORC1-MYC, and AtMORC6-MYC nuclei, respectively. (E) Untransformed wt nucleus subjected to the same antibody staining and imaging procedure. Left panels = anti-MYC channel; middle panels = DAPI channel (gray scaled). DAPI stains DNA, defining the position of dense chromocenters as high intensity white foci; right panels = merged channels (DAPI in blue, MYC in green). White triangles indicate examples of chromocenter adjacent AtMORC localization. Scale bars = 5 μM.

Leaves from three-week old plants were fixed in 4% paraformaldehyde in TRIS buffer (10 mM TRIS pH 7.5, 10 mM EDTA, and 100 mM NaCl) for 20 minutes and washed twice in TRIS buffer. Leaves were chopped in 200–400 microliters lysis buffer (15 mM TRIS pH 7.5, 2 mM EDTA, 0.5 mM spermine, 80 mM KCl, 20 mM NaCl, and 0.1% Triton X-100) and filtered through a 3 μM cell strainer. 5 μL of nuclei suspension was added to 12 μL of sorting buffer (100mM TRIS pH 7.5, 50mM KCl, 2mM MgCl2, 0.05% Tween-20, and 20.5% sucrose) and air dried on chloroform dipped microscope slides for two hours and then post-fixed in 4% paraformaldehyde in PBS for 20 minutes. Slides were washed three times in PBS and incubated in blocking buffer (3% BSA, and 10% horse serum in PBS) for 30 minutes at 37°C. Nuclei were incubated at 4°C overnight in mouse monoclonal antibody against c-Myc (9E10, ab32; 1/200). Slides were washed in PBS and incubated with goat anti-mouse FITC antibody (ab7064; 1/200) for 90 minutes at room temperature. Following PBS washes, nuclei were counterstained and mounted in Vectashield mounting media with DAPI. Nuclei were analyzed with a Zeiss LSM 710 Confocal microscope at 63X or 100X magnification using Zen software.

RPE1 cells grown on coverslips were transfected with βarr2-myc, grown in low serum and then fixed and stained for Kif3A (red) and ab32 (green). Insets show higher magnifications of a representative PC. Kif3A was found in the cytoplasm and at the tip of the axoneme where it was colocalized with βarr2.

Cells were incubated with primary antibodies in permeabilization buffer (PBS with 1 mg/mL bovine serum albumin (PBS-BSA) and 0.1% triton-X-100) for 45 minutes at room temperature. After two washes with PBS-BSA, cells were incubated for 30 minutes at room temperature in PBS-BSA containing secondary antibodies. After one wash with PBS-BSA and two washes in PBS, cells were laid down on microscope slides in a PBS–glycerol mix (50/50) with DAPI.