bEnd.3 cells were grown to confluence in 96-well plates. Wells were decellularized to leave bEnd.3 ECM, as described above. Next, 1000 U87 cells per well were replated onto the U87 ECM and allowed to attach overnight. Wells were washed once with Opti-MEM and then incubated with a dilution series of P1C10- or RBC36-targeted doxorubicin-loaded liposomes. Liposomes were applied to the cell-ECM mixture in a dilution series from 80 μM to 0.3 nM, using fourfold dilutions (concentration calculated from doxorubicin 480-nm absorbance signal), using Opti-MEM as a diluent and incubated for 2 hours. Wells were washed two times with complete media and incubated for an additional 72 hours. CellTiter-Glo (Promega) was used to quantify the number of live cells, according to the manufacturer’s protocol. EC50 calculations were made using GraphPad software suite. To directly quantify VLR-doxorubicin binding to murine sections, P1C10-RBC36, or nontargeted doxorubicin-loaded liposomes were incubated with murine sections, washed and then imaged on a Zeiss Imager Z2 upright fluorescent microscope. A minimum of three fields per group were quantified to determine the VLR channel mean pixel intensity using ImageJ. Data are presented as mean pixel intensity ± SD.
Imager z2 upright fluorescent microscope
Manufactured by Zeiss
The Imager Z2 is an upright fluorescent microscope manufactured by Zeiss. It is designed for high-resolution imaging of fluorescently labeled samples. The microscope features a motorized focusing mechanism and supports a range of fluorescence imaging techniques.
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3 protocols using imager z2 upright fluorescent microscope
1
VLR-targeted doxorubicin uptake in glioblastoma
2
In situ Proximity Ligation Assay for ErbB2 Interactions
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In situ PLA was performed as described.43 (link) HCAEC were seeded onto glass coverslips and allowed to grow to 90% confluence. After appropriate treatments, the cells were fixed with 3% paraformaldehyde prepared in PBS for 10 minutes at 37 °C. In case of cardiac tissue specimens, deparaffinized heart sections were permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature, blocked with 5% donkey serum and 3% BSA in PBS for 1 hour at room temperature, and incubated with primary antibodies in 50% Da Vinci Green antibody diluent (Abcam, Cambridge, MA). PLA was performed following the supplier’s instruction using Duolink Anti-Rabbit PLUS and anti-mouse MINUS PLA probes and Duolink green detection reagent (Duolink, Sigma). To mark the border of the cells, they were stained with goat anti-VE-cadherin antibodies followed by Alexa Fluor 594-conjugated secondary antibodies. Thirty to thirty-five fluorescent images along the z axis with 80 nm intervals (optical sections) were obtained using Zeiss Axio Imager Z2 upright fluorescent microscope via a 63×/1.40 NA objective and deconvolved using AxioVision 4.9 software. PLA reaction products appear as green foci, which originate from the location of ErbB2 interaction with its partners. The number of green foci was counted using Adobe Photoshop.
3
Murine Tissue Immunofluorescence Technique
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Murine immunoflourescence techniques were used in two formats. First, VLRs directly reduced off of yeast surface were reoxidized and incubated with murine tissue sections, snap-frozen, and cut on a cryostat for 1 hour at room temperature. Sections were washed three times with PBS + 1% BSA, 1.5% goat serum and incubated with a master mix containing anti–c-myc (rabbit) antibody (1:500; BioLegend), goat anti-rabbit AF555 antibody (1:1000; Life Technologies), and Isolectin GS-IB4 AF488 (1:400; Thermo Fisher Scientific) for 1 hour at room temperature. Sections were incubated with Hoechst 33342 (1:800) for the final 15 min of labeling. Sections were washed three times, fixed with 4% paraformaldehyde, and mounted for imaging. The second application was identical to the first except VLRs are directly labeled with Cy5 using intein EPL described above (22 (link)). Images were collected on a Zeiss Imager Z2 upright fluorescent microscope. For quantification, a minimum of three fields per group were quantified to determine VLR channel mean pixel intensity using ImageJ. Data are presented as mean pixel intensity ± SD.
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