Dako envision

Tissues were harvested, fixed in 4% paraformaldehyde (pH 7.4) for 24 h at 4 °C and subsequently embedded in paraffin. Immunohistochemical analysis was performed on BAT and WAT sections rabbit anti-UCP1 polyclonal antibody (ab23841; Abcam, Cambridge, UK). Sections were rinsed thoroughly and incubated with labelled polymer HRP anti-rabbit (Dako Envision™+; Dako, Hamburg, Germany) for 1 h. Visualization was performed with 3,3′-diaminobenzidine. Microscopic examination was performed using an Axio Observer Microscop (Carl Zeiss, Jena, Germany). Images were obtained using ZEN2012 software (Carl Zeiss, Germany).

The presence of complement proteins was investigated using immunohistochemistry for C1q, C4d, membrane attack complex (MAC, SC5b-9), and mannose-binding lectin (MBL). Detailed protocols are reported in Table S2. In brief, sections were deparaffinized and antigen retrieval was performed. After blocking for endogenous peroxidase, the sections were incubated with the primary antibody. Binding of the primary antibody was visualized with the appropriate secondary antibody (Dako Envision+, DakoCytomation, Glostrup, Denmark) and diaminobenzidine as a chromogen. Isotype-specific controls were used as negative controls (Table S2). Sections were counterstained with hematoxylin.

Fixed frozen sections of spinal cord from mSOD1^G93A mice (10 μm thick) were prepared as previously described^{32 (link)}. Hypoxia-induced factor 1-alpha (HIF-1α) expression was evaluated in paraffin-embedded 6-μm–thick sections of the lumbar cord from ALS patients and 10 μm–thick fixed frozen sections of spinal cord of mSOD1^G93A mice as previously described^{41 (link)}. The following primary antibodies were used: rabbit anti–HIF-1α polyclonal antibody (1:100; Novus Biologicals), rabbit anti–Iba1 polyclonal antibody (1:1000, Wako) and Alexa Fluor 488®–conjugated mouse anti–glial fibrillary acidic protein (GFAP) monoclonal antibody (1:200; Cell Signaling Technology). The following secondary antibodies were used: AlexaFlour488-conjugated goat anti–rabbit IgG antibody (1:500, Invitrogen) and goat anti–rabbit immunoglobulins conjugated to peroxidase-labeled dextran polymer (Dako Envision+, Dako). For HIF-1α staining, reaction products were visualized with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories). The relative optical densities of HIF-1α immunoreactivity were quantified using ImageJ. The fluorescently labeled sections were examined using an LSM 510 confocal microscope (Zeiss).

Clinical profiles of patients examined in the present study are shown in Supplementary Table 1. Samples from the lumbar cord (L5) were fixed in 10% buffered formalin. For immunohistochemistry (IHC), 6-µm sections were prepared. Deparaffinized sections were incubated 30 min with 0.3% hydrogen peroxide to quench endogenous peroxidase activity and then washed with PBS. The primary antibody was a mouse monoclonal antibody against PIWIL1 (1:500). Samples were autoclaved 15 min before incubation with antibody. Secondary antibody was goat anti-mouse immunoglobulin conjugated to peroxidase-labeled dextran polymer (Dako Envision + , Dako). Reaction products were visualized with 3,3'-diaminobenzidine tetrahydrochloride (ImmPACT DAB, Vector Laboratories), and hematoxylin was used to counterstain cell nuclei. For double IHC, two primary antibodies were combined, including antibodies against TDP-43 (1:1000, rabbit polyclonal, Proteintech) and PIWIL1 (1:500). Alexa Fluor® 488 goat anti-mouse IgG (H + L) antibody (A-11008, Thermo Fisher Scientific) and Alexa Fluor® 568 goat anti-rabbit IgG (H + L) antibody (A-11004, Thermo Fisher Scientific) were used as secondary antibodies. Sudan Black B treatment was performed to reduce autofluorescence from lipofuscin. Images were obtained using an all-in-one fluorescence microscope (BZ-X710, Keyence).