Ab32362

The injured muscles were cut to an appropriate size and fixed in 10% neutral buffered formalin for 1 d. For hematoxylin and eosin (H&E) staining, sections were rinsed with PBS and stained with hematoxylin for 40 min. The sections were subsequently rinsed under running tap water and stained with eosin for 10 min. For immunohistochemistry, slides were deparaffinized, treated with 3% H2O2 for 30 min, and blocked with 2% bovine serum albumin (BSA) for 60 min. The slides were incubated overnight at 4℃ with the primary rabbit monoclonal antibody (Abcam; ab32362; 1:1000 dilution). For antigen retrieval, sections were immunostained using the VECTASTAIN® ABC kit (UNIVERSAL, VECTOR, USA) following the manufacturer's specifications. Diaminobenzidine (DAB) was used for staining, and the sections were counterstained with hematoxylin.

Quadriceps muscles were collected, fixed in 4% paraformaldehyde (PFA), and dehydrated in 30% (wt/vol) sucrose (in PBS). Cryosections that were 14 μm thick were permeabilized in acetone, blocked with 5% bovine serum albumin (BSA) for 1 h and immunolabeled with antibodies against desmin (ab32362; Abcam, Cambridge, UK), CX₃CR1 (ab31331; Abcam, Cambridge, UK), CD68 (MCA1957GA; Bio-Rad, Gladesville, NSW, Australia), CD11b (M1/70; BD Biosciences, San Jose, CA, USA), or Ly6C (HK1.4; Biolegend, San Diego, CA, USA), and detected using Alexa Fluor 488-conjugated anti-rat and Alexa Fluor 568-conjugated anti-rabbit (Thermo Fisher, Australia). Sections were counterstained with Alexa Fluor 647-conjugated phalloidin (Thermo Fisher, Australia), Hoechst 33258 (Sigma-Aldrich USA, Inc.), and mounted with Prolong Gold Antifade (Thermo Fisher, Australia). Images were acquired on an Olympus FV1000 and FV3000 confocal microscope and processed using Imaris 9.2 (Bitplane). Three-dimensional (3D) thresholding analysis of desmin-positive myofibers was performed using Imaris Surfaces function. Desmin-positive and phalloidin-positive myofibers were rendered as surface objects, thresholds were applied using the automated threshold function, and voxel area data were plotted for analysis. Data were generated from ten 30-μm-thick cryosections per group (four mice per group).

For ANP/desmin double immunofluorescence reaction paraffin-embedded tissue sections were deparaffinized and dehydrated. Then sections were treated with 0.01 M citrate retrieval solution (pH 6.0) at 90 °C for 20 min, cooled to room temperature, washed twice in PBS and incubated with 5% bovine serum albumin (SAFC A3059) and 0.5% Triton X-100 (Sigma-Aldrich, T9284) in PBS for 60 min to block non-specific binding sites. Then sections rinsed in PBS and incubated with a cocktail (1:1) of primary antibodies for 90 min at 37 °C: antibody to the Atrial Natriuretic Peptide, mouse monoclonal,1:300 (Millipore, CBL 66, clone 23/1) and desmin, rabbit monoclonal 1:500 (ab32362, Abcam, UK). After washing in PBS sections were incubated with a cocktail (1:1) of two secondary antibodies conjugated with fluorochromes: Alexa 488, goat anti-rabbit 1:300 (A11008, Invitrogen Molecular Probes, USA) and Alexa 546, goat anti-mouse 1:300 (A11003, Invitrogen Molecular Probes, USA). The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Germany). Immunofluorescence-stained preparations were embedded in Immu-mount (Thermo-Shandon) and examined using confocal laser microscope Leica TCS SPE (Leica Microsystems Gmbh, Austria).

For immunostaining, samples were fixed in 4% PFA overnight, embedded in optimal cutting temperature compound (Leica, #4583) and cut at 10 μm per section with a Leica CM1860 microtome (Leica). Sections were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: α-actinin (1:400, Sigma-Aldrich, A5044), N-cadherin (1:500, Sigma-Aldrich, SAB5700640). After washing in 0.25% TritonX-100 in PBS, sections were incubated with fluorescent-labeled antibodies (Molecular Probes, Invitrogen) for 2 h.
For cell staining, cultured myoblasts and myotubes were washed briefly with PBS and fixed in 4% paraformaldehyde for 20 min and then permeabilized with PBS containing 0.125% Triton X-100 for 30 min. After being washed 3 times with PBS, the cells were blocked with 5% BSA at room temperature for 30 min. The primary antibodies were applied at 4 °C overnight. The following primary antibodies were used: desmin (1: 50; Abcam, ab32362), Myosin (1: 100; Abcam, ab264490), Talin (1:50, Abcam, ab71333), and Vinculin (1:500, Sigma-Aldrich, V4139). After washing in 0.125% TritonX-100 in PBS, cells were incubated with fluorescent-labeled antibodies (Molecular Probes, Invitrogen) for 2 h and then visualized under a fluorescence microscope (Leica DM6000).

Macrophages were obtained from bone marrow precursor cells extracted from 4 distinct mice that were cultured in DMEM containing 20% FBS and 30% conditioned medium of L929 cell line (enriched in CSF-1) for 7 days. Macrophages were activated with hrANXA1 for 3 days (10 nM) in DMEM containing 10% FBS. After the washing steps, serum-free DMEM was added for 24 hours to obtain macrophage-conditioned medium. Murine myogenic precursor cells (MPCs) were obtained from TA muscle isolated from 4 mice and cultured using standard conditions in DMEM/F12 medium (Gibco Life Technologies) containing 20% heat-inactivated FBS and 2% G/Ultroser (Pall Inc.). MPCs were seeded at 30,000 cells/cm² on Matrigel (diluted 1:10) and incubated for 3 days with conditioned medium containing 2% heat-inactivated horse serum. In the case of direct treatment of MPCs, cells were directly incubated with 10 nM hrANXA1 for 3 days in the presence of 2% heat-inactivated horse serum. Cells were then incubated with an anti-desmin antibody (ab32362, Abcam), followed by a Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc.) (13 (link), 29 (link)). Cells were stained with Hoechst (Sigma-Aldrich) and mounted in Fluoromount (Interchim), and pictures were taken on Axio Observer.Z1 (Zeiss) at ×20 magnification connected to a CoolSNAP HQ2 CCD Camera (Photometrics).

Antibodies against COL1A1 (ab34710), COL4A1 (ab6586), α-SMA (ab5694/ab7817), vimentin (ab92547), p75^NTR (ab52987), desmin (ab32362), xCT (ab175186), Ptgs2 (ab179800), GPx4 (ab125066), FTH1 (ab65080/ab183781), FTL (ab69090), Atg5 (ab108327), Atg7 (ab133528), LC3B (ab48394), and 4-HNE (ab46544) were purchased from Abcam Technology (Abcam, Cambridge, UK). Antibodies against FTH1 (sc-376594), FTL (sc-390558), and ubiquitin (sc-8017 AC) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against α-SMA (14395-1-AP), Ptgs2 (66351-1-Ig), BECN1 (11306-1-AP), p97/VCP (10736-1-AP), and SQSTM1/p62 (18420-1-AP) were purchased from Proteintech (Proteintech, IL, USA). Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-mouse IgG light chain (AS062), HRP-conjugated goat anti-mouse IgG heavy chain (AS064), and anti-NCOA4 (A5695) were purchased from ABclonal (ABclonal, Wuhan, China). Antibodies against S403-pp62 (#39786), LC3B (#83506), PCNA (#13110), and GAPDH (#5174) were purchased from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA). Monoclonal Anti-FLAG® M2 (F1804), Monoclonal Anti-HA (H9658), and anti-ACTB (A5441) were purchased from Sigma–Aldrich (Sigma–Aldrich, St. Louis, MO, USA).

Muscle samples or HEK293 cell lysates were homogenized in RIPA buffer (50 mM tris, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) supplemented with protease and phosphatase inhibitors (Roche, Indianapolis, IN, USA). Equal protein concentrations were separated on 12% SDS-PAGE gels and then transferred to nitrocellulose membranes. The membranes were blocked in 5% milk TBST (20 mM tris–HCl, 150 mM NaCl, pH 7.6) containing 0.1% (v/v) tween 20 at room temperature for 1 h. Membranes were probed with primary antibodies in blocking solution overnight at 4 °C. Membranes were labeled with the appropriate HRP-conjugated secondary antibodies and then visualized using enhanced chemiluminescence detection reagents (Millipore, Billerica, MA, USA). The following primary antibodies were used: DNAJB6 (ab198995, Abcam), desmin (ab32362, Abcam), TDP-43 (10782-2-AP, Proteintech), LC3B (#3868, Cell Signaling Technology), p62 (ab109012, Abcam), M2 (#14793, Cell Signaling Technology), GFP (#2555, Cell Signaling Technology), β-actin (#3700, Cell Signaling Technology), and GAPDH (#5174, Cell Signaling Technology). Western blots were quantified using ImageJ software, as described previously [33 (link)]. All Western blot analyses were analyzed using biological triplicates (n = 3).