Slowfade diamond

For immunostaining, free-floating sections were permeabilized with 0.1% Triton-X in 0.1 M PBS for 20 min and were then blocked in primary blocking buffer (10% normal goat serum, 2% bovine serum albumin, 0.05% Triton-X) for 30 min. A second blocking step was performed using 0.125 mg/mL goat F(ab) against mouse IgG diluted in primary blocking buffer to block endogenous IgG and Fc receptors. Sections were incubated overnight in indicated antibodies (1:500 BK L6/60, 1:500 HA.11, 1:1000 calbindin). Samples were washed 3× 10 min in PBS containing 0.05% Triton-X and incubated for 1–2 h in 4 μg/mL secondary antibodies (Alexa568-conjugated goat anti-mouse, Alexa488-conjugated goat anti-rabbit for single stains. Alexa568-conjugated anti-mouse IgG2a and Alexa633-conjugated anti-mouse IgG1 were used for BKα and HA co-staining). Secondary antibody was washed out 3× 10 min. Samples were incubated for 20 min with 100 nM MG-TCarb or MG-BTau where indicated, concurrently with 0.8–1.6 μM Hoechst 33342. Three, 5 min washes were performed to remove excess dyes. Slices were mounted using SlowFade Diamond (Thermo Fisher) and imaged on a Zeiss 880 laser scanning confocal or Nikon spinning disk confocal (Andor Technologies, Figure 6G).

The plant tissues were transferred into 150 μl of Cas9 reaction buffer placed on microscope slides containing hydrophilic circular windows with a diameter of 15 mm surrounded by a framework of printed hydrophobic black ink (TF0215; Matsunami, Osaka, Japan) and maintained at room temperature for 5 min for blocking. Then, the same volume of Cas9 reaction buffer containing RNP was added to the slide and mixed by pipetting several times. The slides were placed in a moisture chamber and incubated at 26, 37, or 42 °C for 3–5 h or at 4 °C for 16 h. After the incubation, the reaction was stopped by removing the buffer, and the tissues were washed twice in PBS for 10 min at room temperature. To prevent dissociation of the RNP complex, the tissues were post-fixed with 3.0% PFA for 5–10 min at room temperature, and then washed twice in PBS for 5 min at room temperature. The tissues were mounted with an anti-fade mountant, SlowFade Diamond containing 1 μg/ml DAPI (Thermo Fisher Scientific, Waltham, MA, USA). The slides were placed at 4 °C for at least 8 h until the anti-fading solution had penetrated completely into the tissues.

Samples were incubated at 4°C overnight in the following primary antibodies diluted in wash buffer: Rat anti-Elav (DSHB Cat# 7E8A10, RRID: AB_528218) at 1: 500, rabbit anti-pSmad3 (Abcam Cat# ab52903, RRID: AB_882596) at 1: 1000, and chicken anti-GFP (Abcam Cat#ab13960, RRID: AB_300798) at 1: 1000. Samples were then washed and incubated in the following secondary antibodies: Goat anti-rat Alexa Fluor 488 (Thermo Fisher Scientific Cat# A-11006, RRID: AB_2534074) (Supplementary Fig. S2), goat-anti-rat Alexa Fluor 647 (Thermo Fisher Scientific Cat # A-21247, RRID: AB_141778) (Figs 5 and 6), goat anti-chicken Alexa Fluor 488 (Thermo Fisher Scientific Cat# A-11039, RRID: AB_2534096) (Figs 5 and 6), and goat anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific Cat# A-11011, RRID: AB_143157), each at 1: 500 in wash buffer. Samples were washed thoroughly and rinsed in 1XPBS. Brains were mounted in SlowFade Diamond (ThermoFisher Cat#S36972).

For observation by super-resolution microscopy, zygotes were fixed in 3.2% paraformaldehyde solution after immersion in methanol, as described above. Fixed zygotes were blocked with PEMTB and incubated with an anti-GFP (598, Medical and Biological Laboratories, dilution 1:400) and anti-mCherry antibody (M11217, clone 16D7, Thermo Fisher Scientific, dilution 1:100) overnight at 4 °C. After washing with primary antibodies, the samples were incubated with the corresponding secondary antibodies, enclosed in SlowFade Diamond (Thermo Fisher Scientific) with DAPI, and observed using an FV3000-Olympus Super Resolution system (FV3000-OSR, Olympus), which is based on the algorithm developed for spinning disk super-resolution microscopy^{58 (link)}. We used 100× 1.45NA UPLXAPO100XO (Olympus) to obtain images. For the 3D projection and 3D reconstruction in Supplementary Movie 3, noise reduction was performed and then the images were projected to create 3D projection images using Fiji software. The 3D reconstruction was performed using 3D Viewer in Fiji software.

Cells were grown in 6-well plates on sterile cover slips. Following treatment, cells were fixed with 4% paraformaldehyde in PBS at 25 °C for 10 min. They were then transferred to a membrane permeabilization solution (0.2% Triton X-100) for 10 min. Cells were blocked in PBS that contained 1% bovine serum albumin for 1 h. For p62, LC3B, and ubiquitin staining, cells were incubated with anti-p62 (1:400), anti-LC3B (1:200), and anti-ubiquitin (1:400) antibodies at 25 °C for 1.5 h. Subsequently, cells were reacted with appropriate secondary antibodies labeled with Alexa Fluor 488 or 568 (Abcam, 1:1000) for 30 min. After three washes in PBS, cells were mounted using SlowFade Diamond (Thermo Fisher Scientific, S36964). For Hoechst 33342/propidium iodide (PI) staining, cells were seeded in 6-cm dishes 24 h prior to treatment. Following treatment, cells were incubated with 5 µg/ml Hoechst 33342 (Dojindo, H342) and 10 µg/ml PI (Thermo Fisher Scientific, P3566) for 10 min. Cells were then washed three times in PBS and mounted. All images were captured using a confocal laser scanning microscope (Zeiss LSM 710, Göttingen, Germany). Images were processed using LSM software ZEN 2012 (Zeiss).

Endothelial HUVEC cells and epithelial A549 cells were seeded on an 18-mm diameter glass coverslips in a 12-well plate (Thermo Scientific), at a concentration of 200,000 cells/well and they were grown at 37°C with 5% CO₂ until confluence was reached. The confluent cells were then transferred to antibiotic-free growth medium and infected with pneumococci using a multiplicity of infection (MOI) of 50 bacteria per cell. The mixture was incubated at 37°C with 5% CO₂. After washing with PBS, human cells and bacteria were fixed with 4% paraformaldehyde for 10 min at 4°C, followed by blocking with 1% bovine serum albumin for 1 h at room temperature. A rabbit anti-S. pneumoniae antibody (Abcam) was added, in order to visualize attached extracellular bacteria and also proliferating bacteria for 16 h at 4°C. For HUVEC cells, concomitant incubation of a secondary anti-rabbit antibody with 4′,6-Diamidino-2-Phenylindole, dihydrochloride (DAPI, Biotium) and platelet-endothelial cell adhesion molecule-1 (PECAM-1) conjugated with FITC was carried out for 1 h at room temperature. After the final washing steps with PBS, the coverslips were embedded in SlowFade Diamond (Thermo Fisher), sealed with nail polish and stored at 4°C for subsequent imaging. Images were taken on a confocal laser scanning microscope (LSM710, Zeiss).