Fitc conjugated wga

For visualization of specific protein localization in NRCM, cells were stained for anti‐TIP30 (#ab177961, Abcam, 1:100) followed by Anti‐Rabbit IgG Alexa Fluor^® 488 secondary antibody (#4412, NEB, 1:250) and mouse monoclonal anti‐eEF1A1 (#sc‐21758, Sigma, 1:100) followed by Anti‐Mouse IgG Alexa Fluor^® 555 secondary antibody (#4409, NEB, 1:250). A goat polyclonal anti‐PDGFRα (#AF1062, R&D Systems, 1:100) antibody was used to label cardiac fibroblasts. For visualization of specific protein localization in heart tissue sections after AAV9‐TropT‐TIP30 transduction, these were stained for anti‐TIP30 (#ab71752, Abcam, 1:50) followed by Anti‐Rabbit IgG Alexa Fluor^® 555 secondary antibody (#4409, Cell Signaling, 1:200) together with FITC‐conjugated WGA (#L4895, Sigma‐Aldrich). Nuclear staining was performed with VECTASHIELD Mounting Medium (Vector Laboratories) with DAPI. Representative images were acquired using confocal microscopy. Confocal imaging was performed with a TCS SP2 AOBS scan head and an inverted Leica DM IRB microscope equipped with a 63× oil immersion objective. Image analysis was performed using ImageJ.

Mice muscle tissues were frozen in isopentane that had been cooled in liquid nitrogen. Tissue sections were stained with hematoxylin and eosin (Sigma-Aldrich) according to the standard protocol. Wheat germ agglutinin (WGA) staining was performed using FITC-conjugated WGA (Sigma-Aldrich, #L4859). Quantification of cross-sectional area of the myofibers was performed with NIH ImageJ software. Immunofluorescence (IF) stains were conducted as previously described^{58 (link),59 (link)}. The muscle fibers were stained with antibodies directed against MHC1 (BA-D5, catalog AB 2235587; Developmental Studies Hybridoma Bank) or MHC2b (BF-F3, catalog AB 2266724; Developmental Studies Hybridoma Bank). Immunohistochemistry was performed using the UltraSensitiveTM SP (Rabbit) IHC Kit (Maxim) according to the manufacturer’s instructions using TFEB antibodies (A303-673A, Bethyl Laboratories). DAB Staining Kit (DAB-0031, Maxim) was used according to the manufacturer’s instructions for the developing.

To examine the distribution of GlcNAc sugar bound on C. gigas larval tissues, the larvae were stained with FITC-conjugated WGA (Sigma-Aldrich, Rehovot, Israel). The larvae were immersed in FSW containing 10⁻¹⁰ and 10⁻⁴ M GlcNAc solutions for 2 h and subsequently rinsed three times in 1 L FSW, dyed for 5 min in a solution of FITC-WGA diluted in DW at 0.5 mg mL⁻¹. After incubation for 5 min, the larvae were rinsed by filtered seawater; then immersed in 7.5% MgCl₂ solution in a polystyrene 6-well plate and observed using an epifluorescence microscope (Excitation wavelength (437–460 nm (448 nm)); Emission wavelength (472 nm); Nikon Eclipse Ts2, Nikon Corporation, Tokyo, Japan). The use of MgCl₂ has been reported as an effective anesthetic that renders an animal in a complete loss of muscle tone, thus, subjecting it to a relaxed state suitable for physiological observations [60 (link)]. The untreated larvae exposed to WGA-FITC were used as control. To check for autofluorescence, untreated larvae that were not dyed with WGA-FITC were also compared.

The mice were euthanised, and their hearts were isolated and cut into two transverse slices at the mid-level of the papillary muscles. The samples were fixed with 10% formalin and embedded in paraffin. They were then stained with FITC-conjugated wheat germ agglutinin (WGA) and Masson trichrome (MT). The perivascular fibrotic area was measured as described previously^{12 (link),31 (link),32 (link)}. The sections were deparaffinised and incubated with FITC-conjugated WGA (Sigma-Aldrich) diluted 1:100 (10 μg/mL) in 1% BSA/PBS for 60 min while being protected from light. From each group, several sections were randomly photographed with a fluorescence microscope (LSM 510 META, Zeiss, Japan), and the surface areas of 50 cardiomyocytes were measured using ImageJ software. MT-stained perivascular sections were photographed using an Eclipse 80i microscope (Nikon, Japan). The areas of perivascular fibrogenesis were measured using ImageJ software, and the resulting value divided by the total area of the photograph (12,288 pixels) was regarded as the relative vascularised fibrosis area. The entire heart was imaged with a Leica TL5000 Ergo microscope (Leica Microsystems, Japan), and the area of interstitial fibrosis was measured using ImageJ software. The results are represented as the relative fibrosis area (% of total myocardial area).