Fluoromount g medium

Cells were fixed for 20 minutes with 4% paraformaldehyde-PBS, permeabilized with 0.5% Triton X-100 (#X100, Sigma-Aldrich) for 15 minutes and incubated with 5% BSA (Bovine Serum Albumin) in PBS for 1 hour at room temperature. After saturation, cells were incubated overnight at 4°C with anti-TMS1/ASC antibody (ASC) (#ab155970 rabbit, Abcam, 1/200 dilution) or anti-spike (S) antibody (#GTX632604 mouse, Genetex, 1/100 dilution) in PBS-BSA 5%. Then, cells were incubated with appropriate secondary antibody conjugated to Alexa Fluor 488 (#A11034, Invitrogen, 1/500) or Alexa Fluor 546 (#A11030, Invitrogen, 1/500) at room temperature for 1 hour. Nuclei were stained with Hoechst 33342 (#1874027, Invitrogen, 1/1000 dilution) for 30 minutes at room temperature. After three PBS washes, cells were mounted with Fluoromount G medium (Southern biotech). Cells were analyzed and imaged by Leica Dmi8 microscope using a 60X objective.

Quantitative tyrosine nitration detection was performed as previously described45 (link)22 (link). Briefly, slides were deparaffinized, hydrated, incubated with aqueous sodium dithionite solution (10 mM) for 10 min, washed with distilled water and then incubated overnight at 4 °C with an equimolar solution of AlCl₃ and salicylaldehyde (200 mM). Afterwards, the aqueous solution was removed and sections were mounted in Fluoromount G medium (Southern Biotech, Birmingham, AL). Negative and positive internal controls were included. Fluorescence images were obtained with a fluorescence microscope and quantitative analysis of at least six images per sample was performed with Image J 1.44 m software.

Zebrafish tissues were fixed with 4% paraformaldehyde and processed for immunofluorescence staining. Samples were permeabilized with 0.3% Triton X-100 in PBS, blocked with 10% goat serum in PBS, and incubated at 4 °C overnight with the following primary antibodies: mouse anti-Pgp (1:200 dilution, MA5-13,854 Invitrogen), goat anti-GFP (1:1000 dilution, 600–141–215, Rockland). The following day, after washing with PBS for unconjugated antibodies, immunostaining was completed by a 1-h room temperature incubation with secondary antibody (donkey anti-mouse Alexa Fluor 555; 1:1000 dilution; Thermo Fisher Scientific). Tissue sections were mounted with the anti-fade Fluoromount-G medium containing 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Southern Biotechnology). Images were acquired with a Leica DMi8 epifluorescence microscope.

For liver cirrhosis induction, CCl₄ (289116, Sigma) and phenobarbital (Kern Pharma, Barcelona, Spain) or TAA (172502, Sigma) were used. Rats were anesthetized with ketamine (448.00.03, Merial, Barcelona, Spain) and midazolam (841155.9, Normon SA, Madrid, Spain). Collagenase A (10103586001, Roche) was used for tissue digestion. Reagents for cell isolation included HBSS (H8264, Sigma), collagen type 1 rat tail (A10483‐01, GIBCO), DPBS (L0615‐500, Biowest, Barcelona, Spain), iodixanol (D1556, Sigma) and GBSS (G9779, Sigma). For immunocytofluorescence identification, paraformaldehyde (sc‐281694, SantaCruz Biotechnology, Madrid, Spain), triton (×100, Sigma) and fluoromount‐G medium (100‐01, Southern Biotech, Birmingham, AL, USA) were used. SEM preparations required glutaraldehyde (G5882, Sigma) and HMDS (440191, Sigma).

The differentiation status of the primary muscle cells was evaluated after fluorescence staining assay, with the confocal microscope (Olympus FV1000, Tokyo, Japan). After 5 days of culture, cells were fixed in 4% paraformaldehyde in PBS (Life Technologies, Houston, USA; 10 min, RT), and washed with ice-cold PBS. Next, the cells on slides were permeabilised in Triton X-100 (Sigma-Aldrich, St Louis, USA) and washed with PBS (Sigma-Aldrich, St Louis, USA). Finally, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Waltham, USA) and actin filaments with phalloidin-Atto 633 (Sigma-Aldrich, St Louis, USA). After staining, the cells were washed with PBS, and slides were mounted under Fluoromount-G medium (Southern Biotech, Birmingham, USA). On the stained slides, the fusion index was calculated as a ratio of nuclei in the multinucleated myotubes (more than 2 myonuclei) to the total number of nuclei (mononucleated and multinucleated) [51 (link), 52 (link)] in the randomly selected visual fields (n = 8). Next, the relative to control fusion index was calculated from the obtained results, as a ratio of the fusion index, for each group, compared to the control results. The nuclei were counted manually.

Lungs and ileum were fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich) and then dehydrated in 30% sucrose (Sigma-Aldrich) solution for 2 weeks. Organs were embedded in Tissue-Tek® OCT™ (Sakura) and stored at –80°C prior to being sliced. Sections were incubated for 30 minutes in pre-heated antigen retrieval buffer (Citrate buffer 10 mM pH=6). Sections were incubated in TBS-Triton X-100 0.1% and then in blocking solution containing 1% bovine serum albumin (BSA) -10% fetal bovine serum (FBS) -0.1% Triton X-100 in TBS. Primary antibody directed against CXCL5 (Peprotech) was incubated in blocking solution overnight at 4°C. Sections were rinsed three times in TBS and incubated with appropriated secondary antibody. Slides were counterstained using 4′,6-diamidino-2-phenylindole (DAPI) for 10 minutes, rinsed, and coverslips were mounted with Fluoromount-G medium (SouthernBiotech). Images were treated using ImageJ software.

Retinal samples were fixed overnight at 4°C and then immersed in 30% sucrose for an additional night at 4°C, embedded and frozen in OCT compound (Sakura Finetek, Torrance, CA), and cut into 15-μm thick sections using a cryostat as described previously.^{15 (link)} Sections were immunolabeled for SV2 (Developmental Studies Hybridoma Bank, Iowa City, IA) with secondary antibodies conjugated to Alexa Fluor 488 (Life Technologies, Norwalk, CT), followed by propidium iodide (PI; 1 μg/mL; Sigma-Aldrich Corp., St. Louis, MO) to stain nuclei. Labeled sections were covered with Fluoromount-G medium (SouthernBiotech, Birmingham, AL) and preserved under coverslips sealed with nail polish. Sections were examined with a confocal microscope (LSM510; Carl Zeiss Microscopy, Jena, Germany) by scanning 1.0 μm optical sections with a ×63 oil immersion objective. Control sections were processed simultaneously with experimental sections but without primary antibodies.