Atto 647n

Ni²⁺-monoNTA conjugates of Atto550 and Atto647N were purchased from Sigma-Aldrich. trisNTA amine was purchased from Toronto Research Chemicals. NHS ester forms of Alexa Fluor 405 and Alexa Fluor 647 were purchased from Thermo Fisher Scientific. NHS ester form of Cy3B was purchased from GE Healthcare. The amine-NHS coupling reactions were performed according to the well-established protocol described in Refs. 8 (link) and 9 (link). The conjugates were purified over a reversed-phase C18 (Sigma-Aldrich) column by using an HPLC system, verified by MALDI-TOF-MS, and loaded with Ni(II), identically to the steps described in Refs. 8 (link) and 9 (link). After incubation with Ni(II), the conjugates were purified over a 1-ml HiTrap Q HP (GE Healthcare) column and eluted with a 0–2.5 m NaCl gradient using an FPLC system. It is worth noting that Ni²⁺-trisNTA conjugates of Cy3B and Alexa Fluor 647 were eluted at less than 1 m NaCl concentration, whereas the Ni²⁺-trisNTA conjugate of Alexa Fluor 405 required up to ∼1.6 m NaCl for complete elution (Fig. S1). The final yields, volumes, and concentrations for the three Ni²⁺-loaded trisNTA conjugates are summarized in Table S3.

Conjugation of protein or nanobody was performed with an eightfold (laminin; Roche, 11243217001) or fourfold (R2-myc-his; a gift from S. Oliveira) molar excess of NHS ester (ATTO 425, ATTO 647N; ATTO-TEC, Alexa 488, Alexa 594; Sigma-Aldrich) in a minimal volume (<50 µl) of Milli-Q water (MQ). Protein/nanobody and NHS ester were mixed by vortexing and incubated overnight at 4 °C before storing 10-µg aliquots at −20 °C. Fibrinogen pre-conjugated with Alexa Fluor 647 (Invitrogen, F35200) was reconstituted in 0.1 M sodium bicarbonate buffer, pH 8.3, and aliquots were stored at −20 °C.

Rodent and mouse neonatal cardiomyocytes were incubated with wheat germ agglutinin (WGA) at 37 °C on ice for 60 min and/or mitotracker for 10 min at 37 °C. Cells were fixed with 4% (w/v) PFA and permeabilized with 0.5% (v/v) Triton-X-100. Neonatal cardiomyocytes were incubated with anti-ATP synthase (mouse, Abcam; ab14748), and anti-BK_Ca antibodies, overnight at 4 °C. Secondary antibodies conjugated with anti-mouse Alexa-488 (CST; 4408) and anti-rabbit Atto-647N (Sigma-Aldrich; 40839) were added for 60 min at room temperature. To label nuclei, DAPI was added (1:10,000) in the wash solution. Coverslips were mounted with Mowiol®. Cells were imaged with Nikon A1R high-resolution confocal microscopy. The colocalization index was calculated using protein proximity index analysis (4). Images were filtered by custom-built software as described earlier.

Sample preparation for Airyscan confocal immunofluorescence microscopy. Mice 6 weeks of age were transcardially perfused with 80 mM PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl₂, 4% paraformaldehyde). Eyes were enucleated, and after the cornea was removed, they were immersion fixed overnight at 4 ^oC. After removal of lens, the eyecups were flash-frozen in optimal cutting temperature using liquid nitrogen. 8 μm cryosections were collected and stained for rhodamine wheat germ agglutinin (Vector RL-1022) and phalloidin conjugated to Atto647N (Sigma 65906) or cofilin (CST 5175) and phalloidin conjugated to Atto647N (Sigma 65906). The sections were then mounted in Prolong Glass (Invitrogen P36980). Sections were imaged on a Zeiss LSM 880 Airyscan Fast Confocal Microscope using a 63x objective. Z-stacks were first processed in Zeiss ZenBlue software for Airyscan processing, then colour processing and analysis were performed in Fiji/ImageJ. Actin puncta quantification was performed by taking a 0.35 μm ROI around each actin puncta that was at the base of an outer segment, slice by slice, on each z-stack. Then the averaged relative integrated density of three background ROI’s were subtracted from each relative integrated density measurements from the actin ROIs. These measurements were then plotted in Prism software, where Students t-test statistical analysis was performed.

Nucleosomes were fluorescently labeled in situ after Mlh1–Pms1 diffusion trajectories were recorded on the DNA substrates. An anti-HA antibody targeting (Immunology Consultants Laboratory, RHGT-45A-Z) was diluted 100-fold in BSA buffer and injected into the flowcell at 10 nM final concentration for 5 min. Next, 10 nM secondary antibody was injected and incubated for 7 min, then buffer flow was stopped to visualize nucleosomes on double-tethered DNA molecules. We have used anti-rabbit Alexa488 (Life Tech, A-11008) or anti-rabbit ATTO647N (Sigma-Aldrich, 40839-1 ml) for the secondary antibody.