Vectorshield

Single cardiac myocytes were isolated from right atrial tissue by enzymatic digestion (Lancaster et al., 2004 (link)). Cells or 10 μm frozen sections were subjected to immunolabelling as previously described (Jones et al., 2002 ). Details of the primary antibodies utilised are listed in Table 1. FITC-conjugated anti-rabbit or anti-mouse antibodies, as appropriate for the primary antibody (Dako, Denmark) were used to fluorescently tag the primary antibody. Following immunolabelling, myocytes were incubated for 2 h with wheat germ agglutinin (WGA) conjugated to rhodamine, a lectin that binds to N-acetylglucosamine within membranes (Vector, Burlingame, USA) to permit visualisation of membrane morphology.
To prevent photo-bleaching, myocytes were mounted in vectorshield (Vector, USA) and all slides were stored in the dark at 4 °C prior to examination by laser scanning confocal microscopy (Zeiss, Hertfordshire, UK). All fluorescent images were individually collected for each optical slice, using the same microscope and settings for the lasers and detector, from superimposing 6–10 optical slices taken at ≤1 μm intervals at each wavelength. Images of each intercalated disc were analysed by measuring their axes and area using the confocal software (LSM, Zeiss). Analysis of images such as density of label were performed using ImageJ v2.11x (NIH, USA).

PbMNCs and PbMNCs‐QQ were suspended in EGM‐2MV medium containing 5% FBS and depleted hydrocortisone (1 × 10⁶ cells/mL), and were cultured in a human fibronectin‐coated 96‐well Primaria plate (1 × 10⁵ cells/100 μL/well) for 7 days. On day 7, the medium was replaced with fresh medium containing FITC‐labeled Ulex europaeus agglutinin I (FITC‐UEA‐I, 10 μg/mL, Vector Lab., Burlingame, California) and 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindo‐carbocyanine perchlorate‐labeled acetylated low‐density lipoprotein (DiI‐acLDL, 10 μg/mL, Biomedical Tech., Stoughton, Massachusetts). The attached cells were cultured for 4 hours for EPC labeling. The cells were washed with PBS twice and fixed with 4% paraformaldehyde phosphate buffer solution (PFA, Wako, Osaka, Japan) at 4°C for 30 minutes. The wells were then covered by Vector shield with DAPI (Vector) to avoid fluorescence decay and nuclear staining. The plates were kept at 4°C in the dark prior to observation. In each well, three to five micrographs with random fields were acquired using a fluorescence microscope (Keyence, Osaka, Japan). The FITC‐UEA‐1 and DiI‐acLDL double‐positive cells were counted as early EPC.

Cells were stained for the presence of the tight junction protein ZO-1 (O'Rourke et al., 2016 (link)). Cells were fixed with 4% (w/v) formaldehyde then quenched and permeabilized using NH₄Cl/Triton-X100 solution (50 mM/0.2% v/v). Cells were washed twice with phosphate buffered saline pH 7.4 (PBS), blocked with 1% (w/v) bovine serum albumin (BSA) in PBS for 30 min, and incubated with primary antibody (ZO-1, Rabbit mAb, Biolabs, UK), diluted 1:1000 in a blocking buffer at room temperature for 1h. After incubation, cells were washed three times with the wash buffer (PBS with 0.05% Triton-X100) for 5 min each time on a rocking plate, and incubated at room temperature for 1h with the secondary antibody conjugated with AlexaFluor 488 (anti-rabbit IgG, Biolabs) diluted 1:500. Concanavalin A (ConA) conjugated with rhodamine (1:500 Vector labs, UK) was used for visualization of membranes, Alexa-488 conjugated Phalloidin (1:1000) was used for visualization of actin filaments (Abcam) and nuclei were visualized using Hoechst 33342 (ThermoFisher). Counterstains were incubated on the cells for 30 min before washing in PBS and the addition of the mounting medium (Vectorshield, Vector Labs, UK). Slides were imaged using a Zeiss LSM710 or LSM880 confocal microscope equipped with 63×/1.4 oil DIC objective and Zen black software (Zeiss).

Dorsal root ganglia Lumbar (L) 5 and plantar surface of hindpaws were excised and immersion fixed in 4% paraformaldehyde. This was followed by tissue immersion in 30% sucrose for 24 hours 4^°C. Tissue was frozen in OCT and stored at −80^°C until processing. Tissue sections were cut to thickness of 6 μm for dorsal root ganglia and 20 μm for plantar skin. Slides were washed in PBS and was followed by PBST for five minutes prior to incubation in blocking solution (5% FBS and 10% BSA in PBST) for one hour at room temperature. Sections were then washed three times in PBS for five minutes. Primary antibodies were prepared in blocking solution and sections were incubated overnight at 4^°C. Primary antibodies used were: rabbit anti- vascular endothelial growth factor receptor 2 (VEGFR2: 1:100; Cell Signalling) and mouse anti- protein gene product 9.5 (PGP9.5; 1:10; Abcam). Slides were then washed in PBS three times for five minutes per wash. Sections were incubated with secondary antibody (1:1000; anti-rabbit alexafluor 488 or anti-mouse alexafluor 555) in PBST for 90 minutes at room temperature. Slides were washed in PBS and mounted with vectorshield (Vector Laboratories). Images were acquired using a Leica SPE confocal microscope.