Rhodamine red x

FISH targeting bacterial cells was performed using the oligonucleotide probe Eub338 [11 (link)] custom synthesized and 5’ labeled with Rhodamine Red X (Thermo-Fisher Inc., Waltham, MA). Sections were incubated in a hybridization buffer of 0.9 M NaCl, 0.02 M Tris pH 7.5, 0.01% SDS, 20% HiDi formamide [Applied Biosystems] and 2 μM probe at 46°C for six hours. The Eub338 probe is effective over a wide range of hybridization stringencies [12 (link)] and we routinely employ it at the intermediate stringency of 20%. After hybridization, samples were washed at 48°C for 15 minutes in a large excess of wash buffer (0.215 M NaCl, 0.02 M Tris pH 7.5, 0.005 M EDTA). Additional FISH on sections of gnotobiotic colon was carried out in the same way except using three oligonucleotide probes: Eub338 5’ labeled with Alexa 647, Bthe577 specific for Bacteroides thetaiotaomicron [13 (link)] 5’ labeled with Rhodamine Red X and Erec1259 specific for Eubacterium rectale [13 (link)] 5’ labeled with Alexa 594.

Residual frozen tissue from diagnostic human muscle biopsies were obtained from the University of Iowa Wellstone Muscular Dystrophy Specialized Research Center. Cryosections were prepared on glass slides and maintained at −80 °C. Immunostaining was preformed according to a previously published protocol [90 (link)]. In brief, slides were placed in blocking buffer [PBS2+ (130 mM NaCl, 3 mM Na₂HPO₄, 3 mM NaH₂PO₄, 10 mM EGTA), 0.1% Triton-X100, 0.1% BSA] for 1 h at room temperature. Primary antibodies were diluted in blocking buffer and 40 microliters of primary antibody solution were pipetted onto each sample. Samples were covered with parafilm and incubated in the dark in a sealed humidified chamber for one hour at room temperature. Slides were washed in PBS2+ and stained with appropriate secondary antibodies conjugated to Alexa-488, Rhodamine Red-X, or Texas Red-X (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and under the same conditions as the primary antibody. Slides were washed with PBS2+ and a coverslip mounted with 10 microliters of Vectashield with DAPI (Vector Laboratories, Newark, CA, USA). Slides were imaged on a Leica THUNDER Imager Live Cell & 3D Assay imaging system using a 63X objective (Leica Microsystems, Wetzlar, Germany).

Immunofluorescence detection of R. felis in the infected host cells was performed as previously described [83 (link)]. Confluent XTC-2 and Vero cells were infected with an MOI 25. After 72 h, host cells were fixed with 4% paraformaldehyde in cytoskeleton-stabilizing PHEM buffer [84 (link)], permeabilized with 0.1% Triton X-100 in PBS, and blocked with 5% milk in PBS. Cells were then incubated with a rabbit polyclonal primary antibody against Rickettsia conorii (1:100; prepared in our experimental animal facility at the Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia). For the localization of intracellular bacteria, we applied a goat anti-rabbit IgG (H + L) polyclonal secondary antibody conjugated with Rhodamine Red-X (Invitrogen, reference number R6394, lot 1402199, made in the USA). To visualize filamentous actin, coverslips were probed with Alexa Fluor 488-tagged phalloidin (Invitrogen, reference number A12379, lot 1378369, made in the United States). Finally, samples were mounted with Vectashield Medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI and analyzed using a Zeiss LSM 510 META confocal fluorescent microscope. Brightness and contrast were adjusted by LSM Image browser software (Zeiss, Jena, Germany).

The surface morphology of N-CHDM was observed using phase contrast microscope (Carl Zeiss) and scanning electron microscope (SEM; Phenom G2 Pro Desktop). For immunofluorescence staining of ECM components, N-CHDM was prepared on the glass coverslips, fixed in 4 % paraformaldehyde, then washed with PBS more than three times. Subsequently these samples were treated with 0.2 % Triton X-100 solution for 30 min and they were blocked with 3 % bovine serum albumin (BSA). Once primary antibodies against human fibronectin (Santa Cruz, sc-271,098) and type 2 collagen (COL II) (Abcam, ab34712) were prepared in 1 % bovine serum albumin (BSA) solution, while diluted in 1:200, they were separately treated overnight at 4^oC. As the secondary antibodies diluted in 1:500, Alexa fluoro-488 was used for fibronectin and rhodamine red-X (Invitrogen) was applied for COL II. The intensity and distribution of these ECM proteins were confirmed via confocal microscope (LSM700; Carl Zeiss).

IF-FISH experiments were performed as described before (Takai et al. 2003 (link)). The primary antibodies used were 53BP1 (1:1000; rabbit polyclonal; Novus Biologicals 100-304A) and Myc (1:1000; Cell Signaling 9B11). The secondary antibodies used were raised against rabbit or mouse and conjugated with Alexa 488 (Invitrogen) or Rhodamine Red-X (Invitrogen), and diluted 1:1000. Telomere FISH was performed using Alexa 488 or Cy3-OO-TelC-labeled PNA probes (PNA Bio, Inc.). Streptavidin Alexa 488 (1:2000 dilution; Invitrogen S32354, ) was used for detection of biotinylated proteins by IF.

To investigate if silencing miR-214 affected the protein expression of its target genes, immunostaining was performed. Another well-known transcription regulator, transforming growth factor beta 1 (TGFβ 1) is highly expressed in calcified AVs and is known to mediate phenotypic switch (fibroblast to myofibroblast), fibrosis, apoptosis, inflammation and calcification. So the possible regulation and activation of TGFβ 1 by miR-214 was also tested. Frozen sections were fixed in a 1:1 mixture of methanol/acetone for 10 min at room temperature (RT) and then blocked (1 h, at RT) using 10% (v/v) donkey serum in PBS. Immunofluorescence staining was carried out using following antibodies, KLF4 (Novus Biologicals, CO), TGFβ 1 at a concentration of (1: 200) overnight at 4 °C. Secondary staining was performed using appropriate secondary antibody labeled with Rhodamine Red X (Invitrogen, NY) for KLF4 or DyLight 488-conjugated donkey anti-rabbit secondary antibody for TGF β 1 (Jackson Immunoresearch, PA) (1:500). Nuclei were counterstained with DAPI (1: 5000) and the samples were mounted using anti-fade mounting medium and imaged. ImageJ software was used to quantify the expression of the proteins in the immunopositive cells as reported by Thayer et al.54 (link).