Alexa fluor 488 conjugated anti rat

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 immunofluorescence staining, primary antibodies were diluted in 1% PBS-BSA. Frozen sections were incubated with primary antibodies for Agrin (R&D Systems, #AF550), Hu (abcam, #96474), F4/80(BM8) (Invitrogen, #41-4801-82), Col4 (abcam, #236640), Atf4 (Invitrogen, #MA5-32364), Bax1 (SCBT, #sc-7480), Egr1 (Invitrogen, #MA5-15008), IL-18 (Invitrogen, #MA5-47203) and Hif1a (Invitrogen, #PA116601) overnight at 4 °C, followed by secondary antibodies (Alexa Fluor 647 conjugated anti-rabbit IgG; Alexa Fluor 555 conjugated anti-goat IgG; Alexa fluor 488 conjugated anti-rat; Invitrogen) for 1 h. Cell nuclei were visualized by DAPI. Sections were covered with aqueous Poly/Mount (Polyscience Inc.) and examined with a Zeiss LSM 780 laser-scanning confocal microscope (Zeiss). Images were compiled by ImageJ and Adobe Photoshop CS6 software package.