Vectashield hardset mounting medium with dapi h 1500

The 5 µm frozen section was blocked with with 1% bovine serum albumin (A4503, Sigma-Aldrich) for 30 minutes. Then monoclonal anti-mouse α-SMA (A-2547, Sigma-Aldrich) was used at a dilution of 1∶5000 as primary antibody for 1 hour. The goat anti-mouse Rhodamine TRITC (M1061; Leinco Technologies) was used as secondary antibody with a dilution of 1∶500 for 1 hour at room temperature. Between each step, the slides were washed in PBS buffer 5 minutes for 3 times. The slides were rinsed then counterstained and mounted in Vectashield hard set mounting medium with DAPI (H-1500; Vector Laboratories).

The hearts obtained from chagasic mice were cryopreserved in Tissue-Tek (Sakura, Alphen aan den Rijn, The Netherlands), and 10-μm sections were obtained in Leica CM 1850 UV cryostat (Leica Microsystems, Wetzlar, Germany). To evaluate the presence of GFP⁺ cells in the heart, sections were incubated overnight with the following primary antibodies: anti-connexin 43 and troponin T (Santa Cruz Biotechnology, Inc.). On the following day, sections were incubated with Alexa fluor 594-conjugated anti-goat IgG or Alexa fluor 488-conjugated anti-rabbit IgG (Molecular Probes, Carlsbad, CA, USA). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) by using VectaShield Hard Set mounting medium with DAPI H-1500 (Vector Laboratories, Burlingame, CA, USA). Sections were then analyzed by using a FluoView 1000 confocal microscope (Olympus).

Immediately following slicing, sections were incubated for 72 h at 4 C in a blocking solution [KPBS containing 0.3% Triton X-100, 2% normal donkey serum (017-000-121; Jackson ImmunoResearch, West Grove, PA, USA)], with the primary antibody: c-Fos goat primary (1:2,000; sc-52-G; Santa Cruz, Dallas, TX). After rinses in KPBS, tissue was incubated for 1 h in the dark in the blocking solution containing the secondary antibody: Alexa 546 anti-goat (1:200; A11056; Invitrogen, Carlsbad, CA, USA). Following rinses in KPBS, in semidarkness tissue was mounted onto slides (SuperFrost Plus), dried, coverslipped with Vectashield HardSet Mounting Medium with DAPI (H-1500; Vector Labs, Burlingame, CA, USA), and stored at 4 C until analysis. This immunohistochemistry procedure was successfully repeated 8 times in the current study, and has been used successfully numerous additional times within our laboratory.

Immunofluorescence assays were performed as described in [9 (link),53 (link)]. In brief, cells were seeded in chamber slides, washed with PBS, fixed with ice cold acetone, and permeabilized using 0.1% TritonX-100 (Sigma-Aldrich, St. Louis, MO, USA). After washing and blocking, the cells were incubated with a specific PDE6D antibody (1 in 200 dilution; Thermo Fisher Scientific) overnight. On the next day, the cells were incubated with a secondary antibody (Alexa-Fluor 488 anti-rabbit IgG; Thermo Fisher Scientific). Next, rinsing with PBS and mounting with Vectashield Hard Set Mounting Medium with DAPI H-1500 (Vector Laboratories, Burlingame, CA, USA) was performed.

After deparaffinization, the sections were antigen-activated with Histo VT One (Nacalai Tesque) and blocked using Blocking One Histo (Nacalai Tesque). Primary antibody reactions were performed with anti-HPGDS (Cayman Chemicals, Ann Arbor, MI, USA) at a dilution ratio of 1:2000 in Solution A (Toyobo) and anti-Tryptase (ab2378: Abcam), anti-NG2 (sc-80003: Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-CD11b (66519-1-Ig: Proteintech Group, Inc., Rosemont, IL, USA), anti-Troponin T (MAB18742: Bio-Techne Corp., Minneapolis, MN, USA), anti-α-SMA (sc-53142: Santa Cruz Biotechnology, Inc.), and anti-CD31 (sc-376764: Santa Cruz Biotechnology, Inc.) at a dilution ratio of 1:500 in Solution A overnight at 4 °C. Secondary antibodies were reacted with goat anti-rabbit IgG H&L (Alexa Fluor^® 647) (ab150083: Abcam) for anti-HPGDS and goat anti-mouse IgG H&L (Alexa Fluor^® 488) (ab150113: Abcam) for others in Solution A (Toyobo) (dilution 1:1200) for 1 h at room temperature. Sections were mounted with Vectashield Hard Set Mounting Medium with DAPI (H-1500: Vector Laboratories, Inc., Newark, CA, USA). To confirm the specificity of the antibodies, we also verified that no fluorescence was observed in the negative control without the primary antibody.