Alexa fluor 555

Murine liver tissues were embedded in Optimal Cutting Temperature (OCT) medium and stored at −80 °C. Tissue sections were fixed in 4% paraformaldehyde, permeabilized with PBS + 0.1% Triton X-100, blocked with PBS + 8% normal goat serum, and incubated with primary antibodies. Next, tissue sections were washed in PBS, stained with secondary antibodies including DAPI (Life Technologies, 1 µg/ml), and mounted using Dako Fluorescent Mounting Medium. Goat anti-rat or goat anti-rabbit secondary antibodies conjugated to AlexaFluor488 and AlexaFluor555 were used (Abcam, 1:500). Antibodies used for immunostainings of tissue sections are listed in Supplementary Table 2. All tissue sections were imaged using a Leica SP8 confocal microscope and quantified using ImageJ software.

To confirm and provide quantitative data on the reduction of GFAP⁺ astrocytes, cytoskeletal proteins were extracted from the brains of 4-month-old TSG-6^−/− mice and age-matched wild-type mice as described above. Thereafter, 40 μg of total protein was analyzed by Western blotting as described above. GFAP was revealed using anti-GFAP followed by anti-chicken Alexa Fluor 488 (Invitrogen), and the loading control was stained with mouse anti-β-actin (ab6276, Abcam) followed by donkey anti-mouse Alexa Fluor 555.

Guts were dissected and fixed for 30 minutes using a 4% Parafolmadehyde (VWR, USA) solution in 1X PBS. Then, three 15-minute washes in PBS-Triton 0.1% were performed to permeabilize the guts. After this, blocking was done for 30 minutes by adding a solution of 1X PBS -Triton 0.1%-BSA 1%, and the primary rabbit α-PH3 antibodies (ABCAM, UK) were added (1:800 in 1X PBS-Triton 0.1%-BSA 1%) overnight at 4 °C. Following three 15 minute washes in PBS-Triton 0.1%, the samples were exposed to secondary antibodies Alexa Fluor® 555 (ABCAM, UK) (1:1000 in PBS-Triton 0.1%-BSA 1%) for three hours at room temperature. Phalloidin coupled to Alexa Fluor® 647 (ABCAM, UK), was added for one hour at room temperature (1:500 in PBS-Triton 0.1%-BSA 1%). Finally, three final washes in PBS-Triton 0.1% were performed and the guts were mounted on microscope slides in anti-fade medium (Immu-Mount, Thermo Scientific).

Cells were seeded onto 96-well glass-bottomed plates (Cellvis) at 70% confluency. The following day cells were fixed with 4% paraformaldehyde (PFA) (Affymetrix), washed three times in PBS and then permeabilised with 0.2% Triton X-100 (Sigma). Cells were blocked in 2% w/v bovine serum albumin (BSA) in PBS for 30 min before incubation with primary antibodies for 1 h at room temperature. Plates were then washed in PBS before being incubated with secondary antibodies for 1 h at room temperature in the dark. After washing again in PBS, cells were imaged using the Cell Voyager 7000 spinning disk confocal microscope (Yokogawa) using the 60× water objective. Primary antibodies used for immunofluorescence were: rabbit anti-ASO (Ionis 13545) (14 (link)), mouse anti-LAMP-1 (BD Bioscience), mouse anti-EEA-1 (BD Bioscience), Cis-Golgi (Abcam), mouse anti-alpha tubulin (DM1A, Cell Signalling) and rat anti-tubulin (Alexa Fluor® 647, Abcam). Secondary antibodies used were donkey anti-rabbit (Alexa Fluor® 488) and goat anti-mouse (Alexa Fluor® 555) sourced from Abcam. Nuclei were stained with Hoechst (Thermo Fisher).

Cells were seeded onto 96-well treated plates (PerkinElmer) at 20% confluence. Cells were left for 24–72 h before incubation with CellMask™ deep red stain (ThermoFisher) and then imaging live or fixing with 4% paraformaldehyde (PFA) and washed three times in PBS. For fixed cell samples, cells were blocked in 2% w/v bovine serum albumin (BSA) in PBS for 30 min before incubation with primary antibodies for 1 h at room temperature. Cells were imaged using the Operetta spinning disk confocal microscope (PerkinElmer) using the 63× water objective. Primary antibodies used for immunofluorescence were mouse anti-LAMP-1 (BD Bioscience), mouse anti-EEA-1 (BD Bioscience), Cis-Golgi (Abcam), mouse anti-alpha tubulin (DM1A, Cell Signalling) and rat anti-tubulin (Alexa Fluor® 647, Abcam). Secondary antibodies used were donkey anti-rabbit (Alexa Fluor® 488) and goat anti-mouse (Alexa Fluor® 555) sourced from Abcam. Nuclei were stained with Hoechst 33342 (Thermo Fisher).

Immunofluorescence staining was performed on TMAs and whole section slides. Therefore, paraffin sections were deparaffinised and antigens were retrieved with EDTA at pH 8 (PT Module, Lab Vision Thermo Scientific). Slides were blocked using normal horse serum, for 30 min at room temperature (Vector Laboratories). Slides were incubated overnight at 4 °C with a master mix containing the primary antibodies (LAG3, 1:75, Cell Signaling; CD4, mouse monoclonal 4B12, 1:75, Thermo Fisher Scientific; CD3, rat monoclonal CD3-12, 1:50, Abcam; CD8, 1:100, Dako/Agilent). Slides were washed and stained with a master mix containing the corresponding secondary antibodies coupled to Alexa Fluor 555 (donkey anti-rabbit, Abcam), Alexa Fluor 594 (donkey anti-rat, Jackson Laboratories) and Alexa Fluor 647 (donkey anti-mouse, Jackson Laboratories) for 1 h at room temperature. Nuclei were visualised with DAPI (Sigma-Aldrich). Slides were mounted using an antifade solution (ProLong Diamond, Invitrogen) and scanned with a 40× objective (gSTED super-resolution confocal microscope, Leica). Images were adjusted for brightness and contrast using ImageJ (FIJI).