Normal goat serum (ngs)

To label CARD9 and ASC, stimulated cells were fixed in −20 °C methanol for 5 min, after washing with PBS, non-specific labelling was blocked by incubation at 37 °C for 1 h in 10% normal goat serum (Dako) containing 0.1% saponin (Sigma). ASC was labelled with anti-ASC primary (1:500 dilution, AL177; Enzo) and Alexa-fluor 568 secondary antibody (1:1,000 dilution, anti-rabbit Alexa-fluor 568 (Invitrogen)). To label CARD9 the cells were subsequently blocked in PBS containing 1% BSA and 0.1% saponin. Immuno-labelling was performed with anti-CARD9 (1:300 dilution, sc-49408, Santa Cruz) and anti-Rabbit-Alexa 430 (1:1,000 dilution, Invitrogen). Cells were counterstained using 4,6-diamidino-2-phenylindole mounting medium (Vecta Labs).
Speck enumeration was performed in cells stimulated with nigericin in presence of FAM-FLICA Caspase 8 Assay Kit, green (1:150, Immunochemistry Technologies) and FAM-FLICA Caspase 8 Assay Kit, red (1:150, Immunochemistry Technologies), followed by fixation with 4% paraformaldehyde for 15 min at room temperature. Non-specific labelling was prevented by incubating cells with 10% normal goat serum (Dako) and 0.1% saponin (Sigma) for 1 h. In all, 1,000 cells per treatment were selected from random fields and the specks counted.

Eyes were collected and post-fixed by immersion in 4% PFA for one hour at 4°C. Retinas were dissected, cut radially to allow them to flatten, and post-fixed again for one hour at 4°C. After gentle brushing to remove vitreous body, retinas were incubated overnight at 4°C with Brn3a antibody (1:100, mouse monoclonal sc-8429, Santa Cruz Biotechnologies, Heidelberg, Germany) diluted in blocking buffer (PBS, 2% normal goat serum (Dako, Courtaboeuf, France), 2% triton X100). Retinas were washed three times in 0.5% triton-PBS and incubated with anti-mouse Alexa 488 antibody (1:500, A11-001, Thermofisher scientific, Illkirch, France) in blocking buffer, for 2 hours at room temperature. After two washes in 0.5% triton- PBS, retinas were rinsed in PBS, and then mounted vitreal side up in anti-fading solution (FluoMounting Medium, Dako).

Carotid arteries were cross-sectioned serially (14 µm thick) with the ligation site as the starting point. Sections were rinsed in PBS, permeablized (0.3% Triton X-100 in PBS for 20 min) and blocked for 1 h at RT in blocking buffer (1% BSA/10% normal goat serum (Dako) in PBS). Next, sections were incubated with either primary or isotype-control antibodies overnight at 4 °C, washed three times in PBS, and where needed incubated with secondary antibody for 1 h at RT. All primary and secondary antibodies were diluted using a blocking buffer. For nuclear staining, carotid arteries were incubated with DAPI (1 µg ml⁻¹, 10 min at RT) and then mounted in RapiClear 1.52 for confocal imaging.

Cells were fixed in 100% ice-cold methanol at −20 °C for 10 min, washed three times with 1X PBS and further permeabilized in 0.3% Triton X-100 for 10 min. Following blocking in 10% normal goat serum (MP Biomedicals), cells were incubated with the primary antibody in blocking buffer at 4 °C overnight. The following antibodies were used for immunofluorescence: H4/H2AS1ph (ab177309, Abcam; 1:2000), Lamin A/C (ab238303, Abcam; 1:1000), H3K4me3 (ab8580, Abcam; 1 μg/ml) and H3K36me3 (ab9050, Abcam; 1 μg/ml). Next, cells were washed three times with 1X PBS and following incubation with Alexa Fluor 568 goat anti-rabbit (A11011, Thermo Fisher Scientific; 1:1000) and Alexa Fluor 488 goat anti-mouse (A11001, Thermo Fisher Scientific; 1:1000) secondary antibodies diluted in 10% normal goat serum for 1 h at room temperature, nuclei were stained with DAPI (Dako) or Hoechst 33342 (Invitrogen). Samples were imaged on a ZeissAxio Observer.A1 microscope. For confocal and super resolution microscopy imaging was carried out on a ZEISS LSM 900 with Airyscan 2 using Zen blue for acquisition and processing. Airyscan2 images were processed using the default deconvolution settings and histogram stretching, applied when required, was identical between control and treated samples for each channel.

Repaired left sciatic nerve specimens were harvested during week 8, and 0.5 cm midpoint sections of 3 grafts were stained with toluidine blue and Neurofilament-200 (NF-200). The toluidine blue staining step was described previously. The NF-200 staining step was performed as follows. The acellular scaffolds were embedded in Tissue-Tek® OCT Compound (Sakura Finetek, USA), sectioned at a thickness of 10 μm, and captured on poly-L-lysine-coated slides. After blocking with 2% normal goat serum (Dako, Denmark), the sections were incubated with rabbit anti-rat NF-200 (1 : 2000, Sigma-Aldrich, USA) at 4°C overnight; two-step visualization was performed using GTvision immunohistochemical detection kit (Gene Tech; Shanghai) with hematoxylin counterstaining. The structures of the regenerated nerves were subsequently observed using by light microscopy.
The number of nerve fibers (n), myelin thickness (MT), and diameter of myelinated fibers (FD) were observed using toluidine blue staining and TEM mentioned above, and g-ratio (axon to fiber diameter) was obtained from 5 representative fields in TEM images to quantitatively assess the specimens using Image ProPlus Imaging software 4.5 (Media Cybernetics, Marlow, UK). The g-ratio was calculated according to the following formula [3 (link)]: $\begin{array}{c}g\text{-}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}=\frac{\left(\mathrm{F}\mathrm{D}-\mathrm{2}\ast \mathrm{M}\mathrm{T}\right)}{\mathrm{F}\mathrm{D}}.\end{array}$

Details of the procedures used have been described in our previous study [16 (link)]. Briefly, 4 μm sections were deparaffinized in xylene and rehydrated in a descending ethanol series. After antigen retrieval in 10 mM sodium citrate (pH 6.0), endogenous tissue peroxidase activities were quenched by 3% hydrogen peroxide for 20 min followed by blocking with 5% normal goat serum (Dako, Glostrup, Denmark) for 1 hour. The sections were immunostained with rabbit monoclonal anti-P4HB (at 1:100 dilution; Cell Signalling Technology Inc., Danvers, MA, USA), rabbit monoclonal anti-PECAM-1/CD31 (1:50 dilution; Abcam^®, Cambridge, MA, USA), and rabbit monoclonal anti-VEGF antibodies (both at 1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. After incubation with horseradish peroxidase (HRP)-conjugated antibody (Invitrogen-Zymed Laboratories, South San Francisco, CA, USA) at 1:200 dilution, signal was detected using a ready-to-use DAKO EnVision™+ Kit (Dako). Nonspecific immunoglobulin was substituted as negative controls.