Paraffin sections (3-μm thick) were dewaxed with xylene and ethanol. Antigen retrieval was performed by heating the slides in an autoclave at 121°C for 5 min in citrate buffer, pH 6.0. Sections were washed with PBS and incubated for 30 min at room temperature with blocking solution. These sections were further incubated overnight in a moist chamber at 4°C with primary antibodies. The characteristics of the primary antibodies employed in this study were summarized in Table 1 . The sections were subsequently incubated with fluorescence-labeled secondary antibodies (Alexa Fluor 488 anti-rabbit and Alexa Fluor 594 anti-mouse; Invitrogen) for 1 hr at room temperature. The reacted slides were then mounted with mounting medium with DAPI. The dimeric proteins were detected by super-resolution imaging using structured illumination microscopy (Nikon, Tokyo, Japan). The areas of the yellow, green, and red signals were analyzed by Lumina Vision (Mitani Corp, Japan). We then determined the “% of ER dimer (i.e., ERα/α and ERα/β)” according to the following equation: (Yellow area/Yellow + Green + Red areas) × 100 (%).
Structured illumination microscopy
Manufactured by Nikon
Sourced in Japan
Structured illumination microscopy is a type of fluorescence microscopy technique that uses a patterned illumination to improve the resolution of images beyond the diffraction limit of light. It achieves this by utilizing the Moiré effect to extract high-frequency information that would otherwise be undetectable.
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2 protocols using structured illumination microscopy
1
Quantifying Estrogen Receptor Dimerization
2
Membrane-Coated Plant Organelle Interactions
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We sought to characterize the cross-species impact of mammalian cell membrane coating on plant-derived photosynthetic organelle interactions with macrophages and mature tissue cells (chondrocytes). RAW 264.7 mouse macrophages and mouse chondrocytes were used for the cellular uptake experiments. Cells were incubated in 12-well plates (1 × 105 cells per well) and cultured for 1 day. The NTUs were labelled with DiI before coating with LNPs or CM. Then, DiI-labelled NTUs, LNP-NTUs and CM-NTUs were used at a concentration of 2 × 105 NTUs per cell to study the cellular internalization efficiency. RAW 264.7 mouse macrophages were incubated with the NTUs for 6 h. Chondrocytes were incubated with NTUs for 1, 3 and 6 h. The cell samples were washed three times with PBS for 5 min and fixed with 4% polyformaldehyde (PFA) for 20 min. Then, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 20 min at 25 °C to label nuclei. Finally, the cells were observed by laser confocal microscopy (LCM; Nikon) or structured illumination microscopy (Nikon). The DiI fluorescence signal was measured using a Synergy H4 hybrid microplate reader (Bio Tek). A fluorescence-based assay was performed to estimate the numbers of NTUs delivered to each cell.
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