Alexa fluor 488 donkey anti mouse igg

The porcine model of restenosis after stenting was generated as our previously described.²², ²³ After porcine were sacrificed, the coronary arteries were removed from the heart and fixed with 10% formalin, then paraffin embedded and sectioned into 5 μm. The sections were then stained with haematoxylin–eosin, and observed under the microscope. The expression and localization of METTL3 were determined according to a previously described immunofluorescence method.³, ⁴ Slides with tissue sections were dewaxed with paraformaldehyde and gradually hydrated with ethanol. Then, the slides were placed in EDTA repair solution at 100°C for 20 min. After the slides were blocked with blocking buffer (5% bovine serum albumin) at 37°C for 30 min, the primary antibody METTL3 (Proteintech 15,073‐1‐AP, 1:200 dilution) and α‐SMA (Abcam, ab7817, 1:200 dilution) were incubated for overnight at 4°C. Subsequently, the secondary antibodies (Alexa Fluor 568 donkey anti‐Rabbit IgG [H + L; Thermo Fisher Scientific, A10042] for METTL3 and the Alexa Fluor 488 donkey anti‐mouse IgG [H + L; Thermo Fisher Scientific, A21202] for α‐SMA) were incubated for 60 min at 37°C and followed with diamidino‐phenyl‐indole (DAPI) staining in the dark. After washing with PBS, an Olympus light microscope BX53 system was applied for images capture.

PV-IN or PN specificity of DREADDs activation was controlled by co-labelling of PV or αCamKII with mCherry in 30 μm-thick coronal sections as in [22 (link)]. Primary antibody: PV (1:500; Sigma-Aldrich; P3088); αCamKII (1:200; Thermo Fisher; #13-7300). Secondary antibodies: Alexa Fluor 488 donkey antimouse IgG (1:200; Thermo Fisher Scientific; #R37114), NeuroTrace 435/455 (1:200; Thermo Fisher Scientific; #N21479). Images were acquired by confocal laser scanning microscopy (Zeiss LSM 700; Carl Zeiss AG, Feldbach, Switzerland) and analyzed using ImageJ software (https://imagej.nih.gov/ij/).

Cells were cultured on poly-l-lysine coated coverslips until 30–40% confluent. Control HEK293 and HEK293/Kv1.3 cells were fixed (4% paraformaldehyde, 20 min), washed (3 × 1 ml DPBS) and incubated in DPBS (0.05% Triton X100, 10% NGS; 20 min). Cells were washed (DPBS, 1% NGS) Anti-Kv1.3 Clone L23/27, mouse monoclonal (1:500; cat no. 75-009, NeuroMab, Antibodies Incorporated, UK) or Anti-ATPB rabbit polyclonal (1:500; cat no. ab128743, Abcam, UK) antibody were added in 1% NGS and incubated in a humidified chamber overnight at 4 ^oC. Coverslips were washed with DPBS with 1% NGS (3 x 1ml). Alexa Fluor 488 Donkey anti-Mouse IgG (cat no. R37114, ThermoFisher) secondary antibody or HRP goat anti-rabbit IgG H + L secondary antibody (cat no. 926-80011 LI-COR Biosciences UK Ltd, Cambridge, UK) was added (1:2000 in 1% NGS) for 1 h. Cells were washed with DPBS with 1% NGS (3 × 1 ml), and mounted with VECTASHIELD Mounting Medium with DAPI (VECTOR Laboratories Ltd, UK). Mitochondria were imaged following incubation with MitoTracker Red CMXRos (ThermoFisher) (100 nM; 30 min; 37 °C; 5% CO₂). Excitation/emission wavelengths: 579/599 nm. Imaging was perfomed using a Zeiss LSM800 microscope and processed using Zen Lite 64 (Zeiss, UK) and Image J.