The in vitro uptake of mAbs-EV by NET cell lines was confirmed with two- or three-color confocal microscopy [24 (link),36 (link),51 (link),52 (link)]. The cytoplasm and nucleus were stained with BacMam GFP Transduction Control (Green); the nucleus was stained with DAPI (blue); and the Liss Rhod fluorescent dye (red) was used to monitor the uptake of mAbs-EV. An Olympus 1X-81 confocal microscope with a laser scanning head (Olympus IX81, Center Valley, PA, USA) was used to scan the stained NET cells and collect MitoSox images.
81 confocal microscope
Manufactured by Olympus
Sourced in United States, Australia
The 1X-81 is a confocal microscope designed for high-resolution imaging. It utilizes a laser light source and a pinhole aperture to capture in-focus images from a specific depth within a specimen, enabling optical sectioning. The 1X-81 provides clear, detailed images of samples at the cellular and subcellular level.
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5 protocols using 81 confocal microscope
1
Visualizing mAbs-EV Uptake in NET Cells
2
Visualizing mAb and ADC Binding and Internalization in NET Cells
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Confocal microscopy was used to observe the dynamic surface binding and internalization of mAb and ADC in NET cells following our established protocol.(38 (link), 39 ) Specifically, BacMam GFP Transduction Control was used to stain the cytoplasm and nucleus, and the AF647-mAb or AF647-ADC was used to target cells. The stained cells were observed using an Olympus 1X-81 confocal microscope with a laser scanning head (Olympus IX81, Center Valley, PA). The MitoSox images were recorded and analyzed offline via ImageJ software.
3
Visualizing mAb and ADC Binding and Internalization in NET Cells
4
Immunofluorescence of Fibroblast Proteins
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Immunofluorescence was performed according to standard protocols and as previously described (Zakariyah et al. 2012 (link)). Briefly, fibroblasts were seeded in eight-well chamber slides (Thermo Fisher Scientific; catalog # 154534) for 48 h in DMEM media, rinsed with PBS, fixed with 3% paraformaldehyde (PFA) for 10 min, and permeabilized in 0.5% Triton X-100 for 3 min. Cells were blocked with 5% goat serum and incubated with first primary and then secondary antibodies (diluted in PBS). VECTASHIELD Hard Set Mounting Medium containing DAPI (4′,6-diamidino-2-phenylindole) (Vector; catalog # H-1500) was used to counterstain nuclei. The following primary antibodies were used for immunofluorescence: Anti-PDI [RL90] (1:100; Abcam; catalog # ab2792-100), Anti-TGN38 [21-G (1:250; Santa Cruz Biotechnology; catalog # sc-101273), and LF-68 anti-Pro-COL1A1 rabbit serum (1:200, a gift from L.W. Fisher, National Institutes of Health [NIH]) (Fisher et al. 1995 (link)). Goat anti-mouse IgG 488 (Alexa-Fluor, Invitrogen) and goat anti-rabbit IgG 568 (Alexa-Fluor, Invitrogen) were used as secondary antibodies. Images were recorded with an Olympus 1X81 confocal microscope. The overlap of two proteins on each section was analyzed by Mander's coefficient for green (M1) and red (M2) separately. Colocalization of pro-COL1A1 and PDI or pro-COL1A1 and TGN38 was analyzed from two independent experiments.
5
Fluorescence Microscopy Imaging Protocol
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Single stained cells were viewed with a Leica DMLB fluorescent microscope (Leica Microsystems, Sydney, Australia) and photos taken using a SPOT camera (RTKE Diagnostic Instruments Inx, MI, USA). Double stained cells were observed under the Olympus 1x81 confocal microscope and Images acquired using the Olympus Fluorview FV1000 (Olympus Australia Pty Ltd, VIC, Australia).
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