Vectashield vibrance

Cells were processed using the protocol described by Rothkamm et al. (17 (link)). Briefly, cells were grown on sterile glass coverslips inside a 4-well plate at a density of 10⁴/mL. After 48 h, cells were irradiated with a desired dose (i.e., 20 mG or 2 Gy) at ~60–70% confluency alongside sham-irradiated controls. Cells were analyzed for γH2AX foci at 30 min, 4 and 24 h post-exposure. At these time points, cells were fixed for 5 min in phosphate buffered saline (PBS) with formaldehyde (4%), and permeabilized with Triton X-100 (1%) in PBS for 10 min. Subsequently, cells were blocked with bovine serum albumin (1%) for 30 min and immuno-stained for γH2AX (mouse anti-H2AX, 1:500 dilution, BioLegend^®, London, UK) at room temperature for 1 h. Secondary antibody (goat anti-mouse conjugated with Alexa Fluor^® 555, 1:500 dilution, Fisher Scientific™, Loughborough, UK) and DAPI (1:500) were then added and incubated for 30 min at room temperature. Cells were eventually washed (3x5 min) in PBS to remove excess secondary antibodies before mounting with Vectashield Vibrance^® (Vector Laboratories, Kirtlington, UK). Slides were imaged using Nikon Ti-Eclipse fluorescent microscope equipped with NIS-Elements AR software (version 413.05). One thousand foci or 1,000 cells were scored for each condition, whichever came first based on power and sample size calculations.

Antibody staining was performed on eyes that were enucleated and processed by a freeze substitution method in 10 mL of dry-ice chilled 97% methanol + 3% acetic acid for 4 days at −80°C (Sun et al., 2015 (link)) and embedded in paraffin as described above. Afterward 5-micron sections were cut, and slides were soaked for 2 min each in five steps of xylene, an ethanol rehydration series (100, 90, 80, 70, 60, and 50%), and TBS (Corning 46-012-CM). A Sequenza staining system (Thermo Scientific 73310017, 72110017) was used for immunostaining the slides. Slides were incubated at room temperature (RT; ∼23°C) for 30 min in blocking buffer [2.5% normal donkey serum in TBS (Corning 46-012-CM with 0.01% NaAzide)]. Slides were stained for 1 hr at RT, washed twice for 5 min each with TBST (TBS + 0.1% Tween-20; Biorad 1706531), incubated with secondary antibody for RT for 1 hr, washed twice for 5 min each with TBST, counterstained with 2.5 μM Hoechst 33342 in TBS for 10 min, and rinsed once with TBS. Vectashield Vibrance (Vector Labs H-1700) was used to mount the coverslip, and the sections were imaged using an A1R confocal on a Nikon Ti2 microscope. All primary and secondary antibodies used for this study are listed in Table 1.

Following overnight post-fixation in 4% PFA, spinal cords were transferred to 30% sucrose. Once it had sank in the solution, the spinal cord was blocked in optimal cutting temperature (OCT) media and stored at -80 C. Spinal cords were sectioned at 20 µm and cut into a seven-section series to enable staining for multiple markers in serial. Sections were then processed with (1) cresyl violet, or (2) FluoroMyelin, or with primary antibodies against (3) IBA1 (1:300; Wako # 019-19741) and GFAP (1:500; Thermo # 53-9892-82), or (4) NeuN (1:500; Encor #MCA-1B7). Cresyl violet was performed in manner consistent with previous reports [16 (link)] and FluoroMyelin was performed per the manufacturer instructions. For immunohistochemistry, sections were processed with heat induced epitope retrieval, permeabilized (0.4% triton; 15 min) and blocked (3% serum, 0.2% triton; 60 min). Following blocking, primary antibodies were incubated overnight at 4°. After primary incubation and three serial washes with 1 × PBS, secondary antibodies were incubated for two hours at room temperature. Secondary antibodies were washed off with 1 × PBS and coverslips were mounted with VECTASHIELD Vibrance (Vector Laboratories).

Indirect immunofluorescence staining was performed using pre-diluted mouse monoclonal pan-cytokeratin antibody (cat. nr. 760–2595, Roche Ventana, Mannheim, Germany) and 1:100 diluted rabbit polyclonal anti-vimentin, clone SP20 antibody (Linaris, Dossenheim, Germany), 1:200 diluted mouse monoclonal α-SMA antibody (Cat. Nr. A5228, Sigma, Darmstadt, Germany). Secondary anti-mouse and anti-rabbit Alexa Fluor™ 488 or 594 conjugated antibodies for detection of the immunoreaction were purchased from Molecular Probes (Life Technologies, Darmstadt, Germany). All antibodies were diluted as suggested by the manufacturers. The immunofluorescent-stained slides were cell nuclei counterstained by DAPI (Molecular Probes) and covered in Vectashield Vibrance (Vector Laboratories, Burlingame, CA, USA). Immunohistochemical reactions were acquired in TissueFaxs system and quantified using TissueQuest software as published before [26 ].

Sort-purified cDC1s were allowed to adhere to poly-l-lysine-coated coverslips prior to fixation with 4% paraformaldehyde (PFA) for 10 min. Cells were then permeabilized with PBS containing 0.1% Triton-100 for 3 min prior to blocking with PBS containing 2% bovine serum albumin, 5% normal goat serum and 0.05% Tween-20. Cells were incubated overnight at 4 °C in blocking buffer containing anti-EEA1 antibody (3288, C45B10, 1:250; Cell Signaling Technology) followed by Alexa Fluor 488-conjugated anti-rabbit secondary antibody (A11008, 1 μg ml⁻¹; Thermo Fisher Scientific) and Alexa Fluor 568-conjugated phalloidin to detect F-Actin (A12380, 1 U ml⁻¹; Thermo Fisher Scientific). Coverslips were mounted in Vectashield Vibrance (Vector Labs), and images were acquired using a Marianas spinning disk confocal microscope (Intelligent Imaging Innovations) equipped with SoRa (Yokagawa), Prime 95B CMOS camera (Photometrics) and a 1.45 NA 100× oil objective. Images were acquired using Slidebook software (version 6.0.24; 3i) and analysed using Imaris software (version x64 9.5.1; Bitplane).

Deparaffinization, rehydration, and antigen retrieval of tissue sections was performed as with IHC. Tissues were blocked with Animal-Free Blocker R.T.U. for 30 minutes at room temperature followed by incubation with chicken IgY anti-GFP antibodies (Aves Labs; GFP-1020, 1:250 dilution) overnight at 4°C. Tissues were washed with PBS, and secondary staining was performed for 1 hour in the dark at room temperature with Donkey anti-chicken IgY AF488–conjugated antibodies (Jackson Immunoresearch; 703–545–155, 1:1,000). Tissues were counterstained with DAPI, coverslipped with VECTASHIELD Vibrance (Vector) mounting media, and imaged on a Zeiss LSM 880 confocal microscope.