Alexa fluor 596

Raft paraffin sections (5-μm thick) were stained with hematoxylin and eosin (HE), and with safran (HES) where indicated. Immunohistochemistry was performed on paraffin sections using primary antibodies for keratin 10 (MA1-35540, Thermo Scientific), filaggrin (VP-F706, Vector laboratories), HPV18-E2 (provided by Dr. F. Thierry), HPV18-E4 (provided by Dr. J. Doorbar), HPV18-L1, p16 (sc-56330, Santa Cruz), Ki-67 (18-0191Z, AbCys), and CXCL12 (K15C clone, MABC184, EMD Millipore). Bound antibodies were detected using the LSAB+/HRP kit (K0679, Dako) or the AEC+ High Sensitivity Substrate Chromogen Ready-to-Use System (K3461, Dako). For immunofluorescence, staining with the primary antibody for MCM2 (ab31159, Abcam) was followed by staining with a goat anti-rabbit Alexa Fluor 596 (Invitrogen). Tissues were counterstained with DAPI. For the TUNEL assay to detect apoptotic cells, we used the In Situ Cell Death Detection Kit, Fluorescein (11684795910, Roche Diagnostics) according to the manufacturer’s instructions. In situ hybridization was performed with the wide spectrum HPV biotinylated DNA probe sets able to detect 11 types of anogenital HPV (In Situ Hybridization Detection System, K0601, Dako).

Cells were cultured in 24‐well plates covered with glass overnight at 37 °C with 5% CO2 for 24 h, and then treated with the indicated reagents or not. Cells were fixed with methanol and then permeabilized with 0.5% Triton X‐100. After being blocked by 5% donkey serum, the cells were incubated with primary antibodies at 4 °C. Cells were incubated with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 596 (Invitrogen) for 1 h at room temperature. The nuclei were stained with DAPI for 10 min. The images were obtained using a confocal microscope (Zeiss, Germany).

Tumor tissue was fixed in formalin for 24 h, transferred to 70% ethanol, paraffin embedded, and sectioned (5 μm). Sections were rehydrated through xylenes and an ethanol series. Antigen retrieval was performed using DAKO antigen retrieval in a steamer for 30 min and sections were blocked with DAKO protein block. Primary detection of F4/80 (Molecular Probes), Ki-67 (DAKO), and MECA-32 (BD Pharmingen), was performed overnight at 4°C. Alexafluor-488 and Alexafluor-596 (Invitrogen) were used for secondary detection and DAPI as a nuclear counterstain. To quantify, five images (20× fields) were taken per mouse. Quantification of F4/80 and Ki-67 staining was done using the count tool in Adobe Photoshop CS5. Percent positivity of each stain was determined relative to the total number of cells as visualized by the DAPI counterstain. Quantification of MECA-32 staining was measured as percent positive area using Image J (42 ). Hematoxylin and eosin (H&E) and Masson’s Trichrome staining was performed by the Solid Tumor Pathology Core at OSU.

The following polyclonal rabbit antisera directed against the indicated antigens were produced in our lab and have been used before [23 (link), 35 (link)]. The working dilutions for immunoblots (IB) and IF are indicated: VDAC (IB 1:1,000), ATOM40 (IB 1:10,000; IF 1:1,000), cytochrome C (IB 1:1,000), ATOM69 (IB 1:50), LipDH (IB 1:10,000) and CoxIV (IB 1:1,000). Commercially available monoclonal antibodies were used as follows: mouse c-Myc (Invitrogen, 132500; IB 1:2,000; IF 1:50), mouse HA (Enzo Life Sciences AG, CO-MMS-101 R-1000; IB 1:5,000; IF 1:1,000) and mouse EF1a (Merck Millipore, Product No. 05–235; IB 1:10,000). Monoclonal anti-tyrosinated α-tubulin antibody YL1/2 [54 (link)] (IFA 1:500) produced in rat was a generous gift from Prof. Keith Gull, University of Oxford.
Secondary antibodies for IB analysis were IRDye 680LT goat anti-mouse, IRDye 800CW goat anti-rabbit (LI-COR Biosciences, 1:20,000) and horse radish peroxidase-coupled goat anti-mouse and anti-rabbit (Sigma-Aldrich, 1:5,000). Secondary antibodies for IF were goat anti-mouse Alexa Fluor 633, goat anti-mouse Alexa Fluor 596, goat anti-rabbit Alexa Fluor 488 and goat anti-rat Alexa Fluor 488 (all from ThermoFisher Scientific, 1:1000)

DF-1 cells were grown on coverslips in M24 plates and infected with the different recombinant MVA viruses. After 48 h.p.i., cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min. The cells were permeabilized by using 0.1% Triton X-100 in PBS for 5 min. Nonspecific reactivity was blocked after incubation of cells with 20% FBS–PBS for an hour. Sheep and mouse antisera raised against BTV-4 (dilution 1:500) and RVFV (dilution 1:500) were incubated overnight at 4 °C. Specific secondary antibodies conjugated to Alexa fluor 488 (cat. number A11015, Thermofisher) and Alexa fluor 596 (cat. number A11032, Thermofisher) were used at 1:1000 dilution for the assays. Nuclei were visualized by using DAPI. Laser scanning confocal microscopy images were acquired with an inverted Zeiss Axiovert LSM 880 microscope. Images were analyzed with Zen 2.0 (Carl Zeiss) and Fiji (NIH) software packages.

After 48 h, the culture media was removed, cells were fixed for 10 min in 4% PFA at RT and permeabilized with 0.2% Triton‐X 100 in PBS. CH1 mouse monoclonal antibody (anti‐tropomyosin antibody; DSHB, USA) was prepared at a 1 : 50 dilution in 1% BSA in PBS and incubated with the cells overnight at 4 °C. The cells were rinsed with PBS and goat anti‐mouse secondary antibody, Alexa Fluor 596 (1 : 1500; Thermo Fisher Scientific Inc., UK) was incubated with the cells for 40 min at RT. Cells were mounted on glass slides (GeneTex, USA) and stored at 4 °C. Cells were visualized using the DMIRE2 inverted microscope (Leica) and cells positive for CH1 antibody were considered cardiomyocytes. Cardiomyocytes positive for morpholino uptake were included in the study. The assembly of the sarcomere was divided into four stages, according to the degree of myofibrillogenesis. Over 530 cells were analyzed for this study.