Fv3000rs confocal microscope

For the PLA analysis, the protein-protein interaction was identified using a commercially available PLA kit (Duolink; Sigma-Aldrich) as described before (20 (link)). The signal was inspected using the Olympus FV3000RS confocal microscope with a 60x UPLAPO oil objective. PLA images were taken using the z-stack model and the PLA signal was quantified with the cellSens software (Olympus). To assess AR and p-MED1 protein-protein interaction in clinical prostate tumor specimens, the Duolink® In Situ Detection Reagents Brightfield Kit (Sigma- Sigma-Aldrich, Oakville, Ontario) was adapted from the manufacturer's instructions for use on the Ventana DISCOVERY Ultra autostainer. Antigen was retrieved at 91°C for 64 min in CC1. Tissues were incubated for 12 h at room temperature with the following antibody cocktail prepared in 1× TBS: Anti-TRAP220/MED1 (phospho T1457) (ab60950, abcam, 1:300) and Anti-AR/Androgen Receptor Antibody (441) (sc-7305, Santa cruz, 1:50). PLA method was conducted as previously described in Zhang et al. (21 (link)) with the following adaptations to detect AR/p-MED1 interactions: 1 h ligation step and 2 h amplification step. PLA-stained tissue samples were scanned and then scored digitally by a pathologist with Aperio image scope software (Leica Microsystems).

Immunofluorescent sections were visualized and captured using an Olympus FV3000RS confocal microscope. Images represent confocal Z-stack taken with identical laser and detection settings. For human microglial proximity to plaque quantification, Z-stack images were taken at ×40 magnification (10 slices taken with a Z thickness of 1 μm), 4 images per hemisphere (n = 3 GFP WT & RFP TREM2 KO, n = 3 GFP TREM2 KO & RFP WT). Human microglia numbers and locations were detected and quantified through Ku80 (GFP/RFP positive) immunofluorescence using the Olympus cellSense imaging software. Cells located within the 50 µm radius from the center of the closest Amylo-Glo positive aggregate were detected. The distance of the center of the detected cells from the closest edge of the aggregate was then measured. Expression of HLA-DR or CD9 in GFP versus RFP cells in the same region were also quantified in Olympus CellSens 2.3. T-test run on Graphpad Prism 7.

At room temperature, diluted RBC solution (approximately 0.025% hematocrit) was injected into a homemade cell chamber. RBC fluorescence imaging was performed on an Olympus FV3000RS confocal microscope with an IX83 body combined with an FV31-HSU Hybrid Scanning Unit (Tokyo, Japan). Calcium mobilization can then be imaged with laser illumination (Ex 490/Em 525), where 15% power (maximum power: 50 mW) was used in the laser diode. Bright-field imaging was performed simultaneously on the same microscope. Images were illuminated using the silver-coated resonance scanning mirrors at 10 fps and 512 × 512 frame size, controlled with Olympus CellSens Dimension software. Light signals were collected via an Olympus 40 × /0.95 UPLXAPO (Air) objective. Two channels inside the InGaAsP high-sensitivity detector with quantum efficiency > 45% were used to concurrently acquire images of both bright-field light and fluorescent light. The acquisition was performed with averaging every two frames. No significant photobleaching effect was observed during the time interval used in the experiments. All images were then collected with the Olympus FV31S-SW software.

Paraffin-embedded tissue sections were deparaffinized, rehydrated, and blocked with 10% NGS and 5% BSA for 1 hour after antigen retrieval with EDTA (1 mM at pH 8) (100^oC for 30 minutes). The sections were washed with TBST and incubated with anti-ZIP8 (MilliporeSigma, HPA038832) (1:50), MDR1 (Santa Cruz), and E-cadherin (1:100) (BD Transduction Laboratories) overnight at 4°C. The following day, sections were washed with TBST and stained for 1 hour at room temperature with anti-rabbit Alexa Fluor 488 (Life Technologies) (1:100) and anti-mouse Alexa Fluor 568 as the secondary antibodies, while they were protected from light and exposed to counterstains for DAPI. The sections were imaged on the Olympus FV3000RS confocal microscope with the ×40 oil immersion objective as indicated.

For in situ T-tubule imaging^{27 (link)}, intact mouse hearts underwent Langendorff perfusion at room temperature with 0 Ca²⁺ Tyrode solution (in mM, pH 7.4: NaCl 137, glucose 15, HEPES 20, KCl 4.9, MgCl₂ 1.2, NaH₂PO₄ 1.2), containing 2.5 μM MM 4-64 (Enzo Life Sciences, # ENZ-52252), a lipophilic fluorescence indicator of membrane structure, for 20 min. The hearts were then placed in a perfusion chamber mounted on the stage of an Olympus FV3000RS confocal microscope and imaged in situ with ×60 (NA = 1.4) oil immersion lens. The optical pinhole was set to 1 airy disc (<1 μm axial resolution). Excitation for MM4-64 was 488 nm, and emission was 680–780 nm.

The cell apoptosis in xenograft tumors were detected by the one-step TUNEL cell apoptosis detection kit (Beyotime, Nantong, China). Briefly, the slices were blocked with anti-fluorescence quenching blocking solution and then observed under a fluorescent microscope. The excitation wavelength of Cy3 is 550 nm, and the emission wavelength is 570 nm (red fluorescence). Fluorescent images were collected using Olympus FV3000RS confocal microscope, and fluorescent signal was quantified using ImageJ. The apoptosis rate = (the mean number of apoptotic cells in five random fields/total cell count in that five fields) ×100%.