Tyramide signal amplification kit

The following were from commercial sources: Anti-β-actin monoclonal antibody (Sigma, St. Louis, MO). SuperSignal™ West Dura (ThermoFisher Scientific, Rockford, IL). Anti-mouse αSyn monoclonal antibody (Novus Biologicals, Littleton, CO). Anti-mouse αSyn monoclonal antibody (Abcam, Cambridge, MA). Goat anti-mouse Alex 595 (Invitrogen, Grand Island, NY). Goat-anti-mouse IgG-HRP (Cell signaling, Danvers, MA). Tyramide Signal Amplification (TSA) kit (Perkin Elmer, Waltham, MA). C16-, C18- and C24:1-GCs and C8-GC (Avanti Polar Lipids, Inc., Alabaster, AL).

Histological and IHC analysis was performed as previously described (Murko et al. 2013 (link)). Fluorescence labeling was carried out with Tyramide Signal Amplification Kit (PerkinElmer) according to the manufacturer’s instructions. Detailed information about antibody specification including producer and working dilution can be found in Supplementary Table 2.

Tissue samples were fixed overnight in 4% paraformaldehyde and further embedded in paraffin. All stainings were performed on 4-μm sections. Stainings with hematoxylin and eosin (H&E) were carried out according to standard procedures with an ASS1 staining unit (Pathisto). Fluorescence stainings were performed with the DyLight system (ThermoScientific) or the Tyramide Signal Amplification kit (PerkinElmer), according to the manufacturer's instructions. The slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted in ProLong Gold (Invitrogen).
The primary antibody used for immunohistochemistry (IHC) was H3S10ph antibody (catalog number sc-8656; Santa Cruz).

Dewaxing, rehydration, antigen retrieval and blocking of endogenous peroxidase and appropriate serum blocking were performed as described above for immunohistochemistry. To detect the primary antibody, slides were incubated with peroxidase-conjugated secondary antibody for 30 min and visualised using Tyramide signal amplification kit (PerkinElmer, Inc.) at 1:50 for 10 min (both steps at room temperature). Antigen retrieval was performed using microwave treatment prior to adding subsequent primary antibodies. Same detection steps using different fluorophores were performed for each primary antibody. Nuclei were counterstained with Hoechst (Thermo Fisher Scientific) diluted in TBS at 1:2000. Tissue sections were mounted with Permafluor (Lab Vision™, Thermo Scientific), and tiled images of the whole tissue section were captured at × 20 magnification using LSM 780 Confocal microscope (Carl Zeiss Microscopy, GmbH). Primary and secondary antibody details used for single and triple immunofluorescent staining are in Additional File 1: Table S1.

Isolated cardiomyocytes were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Endogenous hydroperoxidase activity was blocked using 1.5% H₂O₂. After blocking with 3% bovine serum albumin plus donkey anti-mouse IgG for 1 h, myocytes were incubated with the following primary antibodies: N20 (1:50, Santa Cruz Biotechnology), anti-flag (1:100, Sigma-Aldrich), anti-Src (1:100, Cell Signaling), anti-α-actinin (1:200, Abcam), anti-Ca_v 1.2 (1:100, GeneTex), or anti-SERCA2a (1:100, Abcam) at 4 °C over night. After extensive washes in phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄), cells were incubated with a fluorescein-conjugated secondary antibody. To amplify the signal of endogenous AC6, cardiomyocytes labeled with the anti-AC6 antibody (N20, Santa Cruz Biotechnology) were incubated with a horseradish peroxidase-conjugated secondary antibody, and the signal was enhanced using a Tyramide Signal Amplification kit (PerkinElmer, MA, USA). Nuclei were stained with DAPI. The fluorescent images were acquired via a laser-scanning confocal microscope (LSM 510 meta, Carl Zeiss, Germany).

The recruitment of the mbPEGQ30 peptide and subsequent immunohistochemistry was performed as described in [53 (link)] with modifications. In brief, mice were perfused and 30 μm free-floating brain sections prepared as described previously [36 (link)]. Sections were washed in phosphate buffered saline (PBS) and treated with 1% sodium borohydride in PBS for 30 min. Borohydride was removed by washing three times in PBS. Sections were permeabilized and blocked by washing three times in PBS with 0.3% triton (PBST) and incubated overnight with 10 nM mbPEGQ30 in PBST at 4 °C. Rigorous washing was performed to remove unbound peptides with PBST over several hours at room temperature. Sections were then washed three times in PBS before signal amplification using Elite ABC reagent (Vector). Sections were washed three times in TI buffer (0.05 M Tris pH 7.4, 0.05 M Imidazole (Sigma) in PBS). Signals were amplified using the Tyramide Signal Amplification kit (Perkin Elmer) according to the manufacturer’s recommendations. The sections were then mounted on glass slides and processed through 70%, 90% and 100% alcohols and xylene before cover-slipping with DPX mountant (Sigma). Histological images were obtained using a Zeiss AxioSkop2 plus microscope fitted with a Zeiss AxioCam HRc colour camera. Images were recorded using Zeiss AxioVision 4.7.