Fluoview fv3000 confocal microscope

For immunostaining, every one in six serial coronal sections containing the hippocampal region were picked up and six to eight sections of each animal were adopted for the assay. After being washed with PBS following the treatment with a blocking solution containing 3% normal goat serum and 0.1% Triton X-100 in PBS for 1 h at room temperature, sections were incubated with primary antibodies overnight at 4°C. For BrdU immunostaining, samples were pretreated with 1 M HCl at 37°C for 30 min before blocking. On the second day, sections were incubated with fluorescent secondary antibodies for 1 h at room temperature. After washing, samples were mounted onto glass slides and images were captured using a Nikon inverted microscope. The numbers of BrdU⁺, BrdU⁺DCX^+, and BrdU⁺NeuN⁺ cells were quantified. The representative images were captured with an Olympus FLUOVIEW FV3000 confocal microscope. The following primary antibodies were used: anti-BrdU (Catalog# ab6326; Abcam), anti-DCX (Catalog# 4604; Cell Signaling Technology), and anti-NeuN (Catalog# MAB377; Millipore). The used secondary antibody included AlexaFluor568 goat anti-rat (Cat# A11077; Thermo Fisher Scientific), AlexaFluor488 goat anti-mouse (Catalog# A11001; Thermo Fisher Scientific), and AlexaFluor568 goat anti-rabbit (Cat#A11036; Thermo Fisher Scientific).

Cytoplasmic Ca²⁺ was measured using Fluo-4 AM (Invitrogen, F14217), a cell-permeant green-fluorescent high-affinity calcium indicator. Cells were seeded at a density of 25,000 cells/well in a 96-well plate. Eight hours after seeding, medium was changed to differentiation medium. After 24 h, cells were treated with 2 μM Fluo-4 AM in MEM without serum and antibiotics for 1 h. Cells were then washed and incubated for 30 min in Hanks’ Balanced Salt Solution. Fluo-4 fluorescence was measured in a microplate reader (Infinite M200 PRO, TECAN) at an excitation wavelength of 480 nm and emission wavelength of 520 nm, respectively. In one set, 2 mM of EGTA, a selective calcium ion chelator, was used to ensure that the fluorescent signal was due to Ca²⁺.
Live-cell imaging for cytoplasmic Ca²⁺ influx was performed using Olympus FLUOVIEW FV3000 confocal microscope (objective: UPlanSApo 60×/1.35 oil ∞/0.17/FN26.5). Two micromolar retinoic acid was added to Neuro-2a cells seeded in a confocal dish just before the image acquisition. Total 750 images were captured at a frame rate of 30 images per second.

Mouse hearts from different developmental stages were collected and routinely fixed in 4% paraformaldehyde (PFA) overnight, dehydrated in gradient ethanol, embedded in paraffin wax, and sectioned into 5 μm slices.
For histological analysis, the paraffin sections of heart tissues were stained with hematoxylin and eosin (H&E) according to a standard protocol.
For immunohistochemistry and immunofluorescence staining, the paraffin sections were dewaxed, rehydrated, and boiled in citrate buffer (pH 6.0) for antigen retrieval. Then, the sections were permeabilized with 0.1% Triton X-100, blocked with 10% goat serum and incubated with the indicated primary antibodies overnight at 4 °C. Afterward, the slides were washed with PBS 3 times, incubated with secondary antibodies for 1–2 h at room temperature and then washed with PBS 3 times. Immunohistochemical detection was performed with a DAB substrate kit (ZSGB-BIO, cat ZLI-9018, China). A FluoView™ FV3000 confocal microscope (Olympus, Japan) was used to observe the immunofluorescence staining.
The primary antibodies used in immunohistochemistry and immunofluorescence staining are listed in Supplementary Table S5.

Imaging was carried out using an Olympus FLUOVIEW FV3000 confocal microscope. The brains were brought into focus with a 10× (0.40 NA) objective lens before switching to 20× (0.75 NA). Images were acquired at 1,024 × 1,024 pixels with a step size of 3.0 micron; extra care was taken in order to not saturate an image. The laser power and the parameters for image acquisition were kept similar between control and treatment groups.
For data analysis, the imaging files were analyzed using the publicly available imaging software, Fiji. First, the split channel was selected to open the individual channels of each image (red and green). Selective slices were combined and collapsed into a single image using SUM slices. Brightness and contrast were adjusted manually and in a similar manner across all treatments when required for better visualization of the image. Background was calculated from the brain region adjacent to the ROI and subtracted from each channel, i.e., (Green channel-Background) and (Red channel-Background) and is plotted as the change in fluorescence intensity (Δ Intensity). For CaMPARI imaging, the resultant ratios of (RFP-Background)/(GFP-Background) were calculated and plotted.

To quantify the relative abundance of misfolded protein aggregates in HeLa cells, we utilized a Proteostat Aggresome detection kit (ENZ-51035-K100, Enzo Life Sciences). The Proteostat aggresome detection assay was performed according to the manufacturer’s instructions. Briefly, cells seeded on glass slides were washed with PBS, fixed with 4% formaldehyde for 30 min at room temperature, permeabilized (0.5% Triton X-100, 3 mM EDTA) for 30 min on ice under gentle shaking, and stained with Proteostat dye (1:20,000 dilution) for 1 h at room temperature. Nuclei were counterstained with DAPI. Cells treated with 10 µM MG132 (proteasome inhibitor) for 16 h were used as a positive control. Samples stained with DAPI only served as a background control for Proteostat quantification. The cells were imaged with an Olympus Fluoview FV3000 confocal microscope with excitation/emission (Proteostat) = 488/632 nm and (DAPI) = 350/435 nm. Signal quantification was performed with Fiji software^{71 (link)}.