Mowiol

Tissue from mice was dissected and fixed in 4% paraformaldehyde in PBS for 4 hours for spinal cords, and 1 hour for sciatic nerves at 4°C before being transferred into 30% sucrose solution over-night at 4°C for cryoprotection. Tissue was embedded in OCT (1:1 OCT and 30% sucrose for sciatic nerve) and using a cryostat, where spinal cord (25 μM) and sciatic nerve (10 μM) sections were collected on polysine-coated slides (Thermo Scientific). For immunohistochemistry, sections were permeabilized in 0.3% Triton X-100 in PBS and then blocked (4% BSA, 0.3% Triton X-100 in PBS) for 30 minutes at room temperature before overnight incubation with primary antibody solution at 4°C: anti-PGK1 (Millipore, ABS787) and mouse anti-S100 (Abcam, AB7852) or mouse anti-NF heavily phosphorylated 200 (convence, SMI-31R). After PBS washes, sections were incubated with secondary antibody solution for 2 hours at room temperature: Alexa Fluor 488 donkey anti-rabbit IgG (1:500, Life Technologies, A-21206), Alexa Fluor 594 donkey anti-mouse IgG (1:500, Life Technologies A21203). Sections were counterstained with DAPI nuclei stain (1:1,000, Life Technologies, D1306) for 10 minutes before being mounted with 10% Mowiol (Polysciences). Images were taken using a Zeiss LSMZ10 confocal microscope.

Semi-thin sections of interscapular BAT were used for standard immunolabeling procedure, using a primary antibody against CAT and an appropriate fluorochrome-conjugated secondary antibody (1:400; Alexa Fluor^® 488 goat anti-rabbit, Thermo Fisher Scientific, Waltham, MA, USA). Sytox orange (1 μL ml⁻¹, Thermo Fisher Scientific, Waltham, MA, USA) was used for nuclei counterstaining. Slides were mounted with Mowiol (Polysciences, Eppelheim, Germany), and confocal images were acquired with a Leica TSC SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) using 63/1.4 NA oil immersion lens. The specificity of immunofluorescence was tested by the omission of the primary antibody.

Sections of paraffin embedded brains were dewaxed in Histoclear™ or Xylene (2 × 5 min). Slides were rehydrated through decreasing ethanol series (2 × 5 min: 100%, 3 min: 95%, 75%, 50%, 30%), washed in water (5 min) and PBS (5 min). Frozen sections were warmed to room temperature and washed in PBS (5 min) to remove OCT. If required, antigen retrieval was carried out by placing sections in antigen retrieval solution (Vector Laboratories) and microwaved until boiling for 20 min, after which they were allowed to cool (20 min). After antigen retrieval, slides were briefly dried, sections circumscribed with ImmEdge PAP pen (Vector laboratories Ltd, UK) and incubated in blocking buffer at room temperature (1 h) followed by incubation with primary antibodies (see Supplementary Material, Table S2 for list of primary antibodies) overnight at 4°C. Slides were washed in PBS with Tween-20 0.1% (v/v) (PBST, 4 × 10 min). Appropriate secondary antibodies (Supplementary Material, Table S2) were used at a dilution of 1:1000 in blocking buffer and incubated for 1 h at RT. In all cases 4′,6-Diamidino-2-Phenylindole (DAPI) was also added to secondary antibody mixture (1 μg/ml). Slides were washed in PBST (2 × 10 min) followed by PBS (2 × 10 min) and coverslips mounted with Mowiol (Polysciences Inc., Germany).

Immunohistochemistry analyses were performed on frozen brain sections by standard indirect staining as in [55 (link)]. Antibodies were diluted in 0.1 M PB containing 1% fetal bovine serum (FBS, Hyclone), 0.06% Triton-X100 (Sigma), and 150 mM glycine (Merck). Rabbit anti-Iba1 (1:100, Wako) was used to detect expression of bMyC; rat anti-mouse I-A/I-E (1:100, clone 2G9, B.D. Pharmingen) was used to detect antigen-presenting cells; mouse anti-phospho-Ser 139-Histone H2AX (clon JBW301, Millipore) was used to analyze DNA damage. Alexa fluor 488-labeled donkey anti-rabbit, -rat, -mouse, and Cy3-labeled donkey anti-rat antibodies (Jackson) were used as secondary antibodies. After staining, all sections and cells were mounted and preserved with 50% Mowiol (Polysciences), 2.5% DABCO (Sigma).

Semi-thin sections of interscapular BAT were used for standard immunolabeling procedure, using primary antibodies against Nrf2, 4-HNE, and appropriate fluorochrome-conjugated secondary antibody (1:400; Alexa Fluor^® 488 goat anti-rabbit for Nrf2, A11008, or Alexa Fluor^® 488 goat anti-mouse for 4-HNE, A11001; Thermo Fisher Scientific, Waltham, MA, USA). DAPI (1 μL mL⁻¹, Sigma-Aldrich, Germany) was used for nuclei counterstaining. Slides were mounted with Mowiol (Polysciences, Eppelheim, Germany), and confocal images were acquired with a Leica TSC SP8 confocal microscope (Leica Microsystems) using 63/1.4 NA oil immersion lens. The specificity of immunofluorescence was tested by the omission of the primary antibody.

Neurons were fixed in 4% paraformaldehyde (PFA) at room temperature. After permeabilization with 0.1% Triton-X/PBS, cells were blocked in blocking buffer (2% glycine, 2% BSA, 0.2% gelatin and 50 mM NH₄Cl) and subsequently probed with primary and secondary antibodies (Additional file 1: Table S1) as described previously [32 (link)]. Coverslips were mounted with Mowiol (# 17951500; Polysciences Inc., Hirschberg an der Bergstraße, Germany).

Generation of the chicken anti‐EAAT2a (zebrafish) antibody is described elsewhere (Niklaus et al., 2017 (link)). Five dpf larvae were fixed in 4% PFA in phosphate buffered saline (PBS, pH 7.4) at room temperature for 40 min. Embryos were cryo‐protected in 30% sucrose in PBS at 4°C overnight, embedded in Tissue Freezing Medium TFMTM (Electron Microscopy Sciences), cryo‐sectioned at 14–16 μm and mounted onto Superfrost slides (Thermo Fisher Scientific). Immunohistochemistry was performed as described before (Niklaus et al., 2017 (link)). Chicken anti‐EAAT2a 1:500, mouse anti‐synaptic vesicle 2 (IgG1, 1:100, DSHB USA), mouse anti‐acetylated tubulin (IgG2b, 1:500, Sigma 7451) and mouse anti–glutamine synthetase (IgG2a, 1:200, EMD Millipore, MAB302) were used as primary antibodies. Secondary antibodies were goat anti‐chicken Alexa Fluor 488, goat anti‐mouse IgG2a Alexa Fluor 568, goat anti‐mouse IgG2b Alexa Fluor 647 and goat anti‐mouse IgG1 Alexa Fluor 647, all 1:500 (all from Invitrogen, Thermo Fisher Scientific). Slides were cover‐slipped using Mowiol (Polysciences) containing DABCO (Sigma‐Aldrich) and imaged with a TCS LSI confocal microscope (Leica Microsystems). Images were adjusted for brightness and contrast using Affinity Photo Version 1.8 and assembled in Affinity Designer Version 1.7.