Alexa fluor 594 goat anti rat

Human muscle satellite cells (hMuSCs; ScienCell Inc., Carlsbad, CA, USA) were cultured in growth media (DMEM containing 10% FBS, 10% HS, 0.5% chick embryo extract (CEE; MP Biomedicals, Santa Ana, CA, USA), and 1% P/S). On the day of cell seeding, hMuSCs were trypsinized and resuspended in low‐serum medium (DMEM containing 10% FBS and 1% P/S) at a concentration of 3 × 10⁶ per mL, and seeded onto the muscle matrix derived from decellularized gastrocnemius muscles. After 3 or 7 days, hMuSC‐ECM cultures were fixed in 2% PFA for 20 min and transferred to 30% sucrose solution for 24 h. The fixed tissues were then snap‐frozen in liquid nitrogen‐cooled 2‐methylbutane. Samples were then cryosectioned (7 μm) and mounted on glass slides. Sections were incubated overnight with either rat anti‐Er‐Tr7 antibody (1:500; Hycult, Plymouth Meeting, PA, USA) or rabbit anti‐desmin antibody (1:1000; Abcam, Cambridge, MA, USA), followed by a 45‐min incubation with AlexaFluor 594 goat anti‐rat or AlexaFluor 488 goat anti‐rabbit secondary antibodies (1:1000; Life Technologies).

These procedures were carried as described previously [28 (link)]. Primary antibodies used were rat monoclonal mCherry (1:2000, Thermo Fisher, M11217); mouse monoclonal TH (1:2000, Sigma, T2928); secondary antibodies were Alexa Fluor 488 goat anti-mouse (1:1000, Invitrogen Molecular Probes, A11001), Alexa Fluor 594 goat anti-rat (1:1000, Invitrogen Molecular Probes, A11007). Slices were mounted on slides, embedded in Mowiol (with 4,6-diamidino-2-phenylindole), cover-slipped, and analyzed using an upright fluorescent microscope (Nikon Eclipse 80i, Nikon).

AT samples were fixed and mounted in paraffin and sectioned into 5 μm slides with a microtome (Leica Biosystems, Nussloch, Germany). Antigen was unmasked with proteinase K (cat#S3020, Dako, Santa Clara, CA) and blocked with 15% horse serum for 1 h. Samples were incubated with the following primary antibodies overnight at 4°C: mouse anti-human CCL17 (1:50, cat#DY364-05, R&D Systems), mouse anti-human CCL22 (1:50, cat#DY336, R&D Systems), goat anti-human CCR4 (1:100, ab1669; Abcam, Cambridge, UK), rabbit anti-human CD3 (1:100, cat#C7930, Sigma-Aldrich, St. Louis, MO), rabbit polyclonal anti-human CD31 (1:50, cat#ab32457, Abcam) and rat anti-human Mac-3 (1:100, cat#sc19991, Santa Cruz Biotechnology, Dallas, TX). Alexa-Fluor^® 488 goat anti-rabbit (cat#A11034), Alexa-Fluor^® 488 donkey anti-rat (cat#A21208), Alexa-Fluor^® 594 goat anti-rat (cat#A1107), Alexa-Fluor^® 594 goat anti-mouse (cat#A1105) and Alexa-Fluor^® 594 chicken anti-goat (cat#A21468) antibodies were used as secondary antibodies (dilution 1:1000; all from Molecular Probes, Eugene, OR). Nuclei were stained with Hoechst (1:4000). Afterwards, five fields from each section were captured with a Zeiss Axio Observer A1 fluorescence microscope (Carl Zeiss Micro Imaging GmbH, Oberkochen, Germany).

The viral expression in the injected mice was immunohistologically validated. Mice were transcardially perfused with PBS, pH 7.4. The brains were removed and fixed overnight in 4% PFA. One hundred μm of free-floating slices were permeabilized in 0.5% Triton X-100 for 1 hr. Subsequently, slices were incubated with a mCherry antibody (1:10,000, rat serum, Thermo Fisher Scientific, M11217) in a blocking reagent (4% normal goat serum, 0.4% Triton 1%, and 4% BSA in PBS) over night at room temperature. After washing the samples three times with PBS, a secondary antibody was administered (Alexa Fluor 594 goat anti-rat, 1:400, Invitrogen, A11007) in 5% normal goat serum/BSA and incubated for 2 hr at room temperature. During the last 30 min of incubation, Nissl staining was carried out (Invitrogen, N21473, 1:200). Afterward slices were washed three times with PBS, mounted with Dako Mounting Medium, and covered with a glass cover-slip. Alternatively, a NeuN staining was performed to visualize neurons in the hippocampus using a NeuN primary antibody (1:1000, MABN140, rabbit, Sigma-Aldrich, secondary Alexa Fluor 405 goat anti-rabbit, A31556, 1:400on).