Bodipy tr methyl ester

Dehydrated embryos or adult eyes were embedded in JB-4 plastic resin (Polysciences) and then sectioned (5 μm) using a LEICA RM 2165 rotary microtome. Sections, in which optic nerve was present, were stained with Hoechst (H3570, 1:2000, Thermo Fisher Scientific) and BODIPY TR Methyl Ester (C34556, 1:200, Thermo Fisher Scientific), and mounted using Permount (SP15-500; Thermo Fisher Scientific). Two-color z series were acquired using a Zeiss LSM5 Pascal confocal microscope via sequential laser excitation with 40× and 63× oil objectives. Images were deconvolved using Huygens Essential software and processed using Adobe Photoshop. Adult eye sections were stained with Methylene Blue/Azure II (Humphrey and Pittman, 1974 (link)) and examined using a Nikon E800 microscope equipped with a Spot Insight charged-couple device digital camera. Quantification of photoreceptor outer segment (OS) length was performed on maximum intensity z projections of confocal stacks created in ImageJ. OSs were traced and measured using the freehand line tool.

For confocal imaging, live embryos and larvae were stained with 100μM BODIPY TR Methyl Ester (Invitrogen) in PBS for 30min, washed 3X in PBS for 5min each, and mounted in 1% low melting agarose with 0.02% MS222.

Zebrafish in vivo skeletal staining was incubated with 0.2% Calcein (Sigma, C0875) solution (pH 7.0) for 10 min or 0.2% Calcein blue (Sigma, M1255) solution (pH 7.0) for 1 h or 0.05% Alizarin red (Sigma, A5533) for 1 h and then washed with system water three times. Cell internal membranes labeled with vital dye BODIPY TR Methyl Ester (C34556, Invitrogen) as described by the manufacturer, the embryos were stained with 100 μM MED for 1 h, then wash three times with egg water. BODIPY TR Ceramide (D7540, Invitrogen) prominent labeling of the Golgi apparatus with 50 μM for 2 h. LysoTracker Green DND-26 (L7526, Invitrogen) was used to in vivo label acidic notochord vacuoles after 5–6 dpf, 50 μM stained for 1–2 h depending on acidification degree of notochord vacuoles. Zebrafish embryos were anesthetized in tricaine (Sigma) and mounted in 1% LMP agarose. Calcein staining embryos were imaged with a SteREO Discovery 20 microscope (Carl Zeiss). Other live staining embryos were imaged with a LSM880NLO confocal microscope with a ×20 water immersion objective (Carl Zeiss). Images collected every 10 min for time series live cell imaging of notochord development from 20 to 30 hpf.

To confirm the intracellular distribution of the compound in tumor cells, MMT1242 was labeled with a nitrobenzoxadiazole frame, which emits green-yellow fluorescence at 552 nm upon excitation at 413 nm, forming NBD1242 (Figure 3). A total of 1.5 × 10⁴ cells/well of CT26 tumor cells were seeded on collagen I-coated 12 mm cover slips within the 24-well plates. The cells were incubated at 37 °C for 24 h before the medium including NBD1242 (3 mM) was added. After incubation for 23 h, the supernatant was aspirated and BODIPY^® TR methyl Ester (Invitrogen, C34556, GFP Counterstain) 5 μM was added at 37 °C for 30 min. After incubation, the solutions were washed by culture medium and 1 μM Hoechst 33,342 (DOJINDO LABORATORIES, H342) stain was added at room temperature for 10 min. The sample solutions were washed by culture medium twice before a PBS 1× wash and fix with 4% paraformaldehyde at room temperature for 20 min. The cells were mounted on slides, incubated 3 times with PBS and ProLong Diamond (Invitrogen, P36961) for 5 min each, then stocked at 4 °C for 4 days. Samples were observed under a KEYENCE BZ-X710 (Keyence, Co. Ltd., Tokyo, Japan) fluorescence microscope.

Zebrafish larvae were fixed in 4% PFA at room temperature (RT) for 30 min, embedded in Neg50 (Richard-Allan Scientific) and cryosectioned following standard protocols. Non-specific binding was blocked using PBDT (PBS, 1% DMSO, 0.5% Triton X-100, 2mg/ml BSA) with 10% goat serum for 30 minutes at RT before incubation with primary antibodies overnight at 4°C. Primary antibodies were rabbit anti-Syntaxin3 (1:400, Alomone labs, ANR-005), rabbit anti-SNAP25 (1:1000, StressGen Biotechnologies, VAP-SV002), mouse monoclonal anti-Rab8a (1:100, Novus Biologicals, clone 3G1), rabbit anti-Cacna1fa (1:5000, a gift from Michael Taylor, University of Wisconsin [75 (link)]), rabbit anti-blue opsin (1:250, gift from David Hyde) and rabbit anti green opsin (1:400, gift from David Hyde, University of Notre Dame). Secondary antibodies were Alexa Fluor-conjugated goat anti-rabbit or goat anti-mouse IgG (1:400, Life Technologies). BODIPY TR methyl ester (1:300, Invitrogen) or Vybrant DiO (1:200, ThermoFischer) was applied for 20 min and nuclei were counterstained with DAPI. Confocal laser scanning microscopy was performed on a Leica HCS LSI or a Leica SP5 microscope. All immunofluorescence experiments were performed at least in duplicate with at least 10 animals per condition.