Vectashield mounting medium for fluorescence

UCA, UCV, and WJ were immersed in optimum cutting temperature (OCT) compound (Leica, Wetzlar, Germany) and frozen at −70°C until sectioning. The tissues were serially sectioned to 6 μm thickness using a cryostat (Leica). Expression of PDGF-Rβ (ab32570, Abcam, Cambridge, UK), NG2 (ab139406, Abcam), α-SMA (ab5694, Abcam), and CD146 (ab75769, Abcam) was detected by immunofluorescence staining. After incubated with primary antibody at 4°C overnight, the frozen sections were then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1 : 200, Invitrogen, Grand Island, NY, USA) or Alexa Fluor 555-conjugated goat anti-rabbit IgG (1 : 200, Invitrogen). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), which was contained in the Vectashield mounting medium for fluorescence (Vector Laboratories Inc.). The images were visualized using fluorescence confocal microscopy (Leica) under a magnification of 600x. The integrated optical density (IOD) values of positive staining in five randomly selected fields of view were tested by Image pro-plus 6.0 software (Media Cybernetic, Rockville, USA).

Cells on coverslips were fixed with 4% paraformaldehyde diluted in PBS solution for 15 minutes, then rinsed with PBS (phosphate buffered saline) for 5, 10, and 15 minutes and stored until staining in PBS. Cells were permeabilized with 0.1% Triton X-100 for 15 minutes then incubated for 1–2 hours at room temperature in blocking buffer (5% Donkey serum +0.5% BSA in PBS) to prevent nonspecific binding. Fixed and permeabilized cells were incubated overnight with primary antibodies (diluted in blocking buffer) at 4 °C. Antibodies used and their concentrations are listed in Table 1. The cells were rinsed with PBS for 5 minutes, 0.01% Trition X-100 for 10 minutes and PBS for 15 minutes, and then subjected to incubation with the secondary antibodies (1:250 diluted in blocking buffer) for 1–2 hours at room temperature. The cells were then rinsed with PBS for 5 minutes, 0.01% Trition X-100 for 10 minutes and PBS for 15 minutes and mounted onto glass slides using ProLong Gold Antifade Mountant with DAPI (Thermo FisherP36931) or Vectashield mounting medium for fluorescence (Vector laboratories, Burlingame, CA) and imaged using a Zeiss LSM 510 confocal microscope.

Irradiated or sham-operated rats were deeply anesthetized and perfused transcranially with cold 0.9% saline, followed by four percent paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) 24 or 48 hours after 60 Gy gamma radiation, n = 6 in each group. The brains were kept in four percent paraformaldehyde in PBS for three days and then cut into 50 μm coronal sections with a vibratome (VT1000S, Leica Microsystems, Wetzlar, Germany). Free-floating sections were washed in PBS and then treated with one percent H₂O₂ for 20 min. Nonspecific binding was prevented by incubating the sections for one hour in five percent normal goat serum in PBS containing 0.3% Triton X-100. The sections were incubated overnight at 4°C with various primary antibodies. After being washed three times in PBS, the sections were then incubated for one hour at room temperature in a relevant secondary antibody solution made up in PBS. The sections were washed and mounted on slides with Vectashield mounting medium for fluorescence containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc. Burlingame, CA) and studied by fluorescence microscopy.

Immunodetection of γH2AX was performed on the same samples previously analyzed for TUNEL or sorted after Annexin-V/PI staining. Pericentrin detection was performed on newly obtained samples of irradiated lymphoblasts allowed to attach onto poly-L-lysine slides. Cells were fixed for 15 min in 4% paraformaldehyde and permeabilized in 1xPBS-0.5% Triton-X100 solution for 15 min. After 30 minutes of blocking with 0.1% Tween20 and 5% FBS, mouse anti-γH2AX (Ser139) (Upstate/Millipore, MA, USA) or rabbit anti-pericentrin (Abcam, UK) was applied at a 1 : 1000 concentration and allowed to incubate for 1 hour at room temperature. Anti-mouse Cy3 (Amersham Biosciences/GE Healthcare, NJ, USA) and anti-rabbit A488 (Invitrogen/Molecular Probes, OR, USA) secondary antibodies were applied at 1 : 1000 final concentration for 45 minutes at room temperature, followed by extensive washing. Before analysis, Vectashield Mounting Medium for fluorescence (Vector Laboratories Inc., CA, USA) supplemented with DAPI was applied. Slides were analyzed using an Olympus BX41TF epifluorescence microscope equipped with an Olympus U-TVIX digital camera using the Isis v5.4.9 software (MetaSystems, Germany).

Pupae were aged to 45-47 h APF. Pupae were dissected, fixed and mounted as in (Wang and Yoder, 2011 (link)). Fixation was in 4% paraformaldehyde with 0.1% Triton X-100. Tissue was washed in PBS before staining with Alexa Fluor 488- or 594-conjugated phalloidin (Molecular Probes) and mounting in Vectashield mounting medium for fluorescence (H-1000, Vector Labs). Z-stacks were captured using a Leica SP5 confocal microscope. Images were processed in Fiji including image stitching using the MosaicJ plugin (Thévenaz and Unser, 2007 (link)).

An immunoblot assay was performed to assess PARP and caspase 3 cleavage for cells in the control and pretreated cultures. Nuclear morphological changes were assessed using Hoechst 33342 dye and visualized under a confocal microscope. For staining assays with the Hoechst 33342 nuclear dye, cells were cultured on glass-bottom dishes. After treatments, the cells were fixed with 3.7% paraformaldehyde for 20 min, washed with PBS three times, and stained with 1 μg/mL solution for 30 min. Stained cells were mounted using Vectashield Mounting Medium for Fluorescence (Vector Laboratories, Burlingame, CA, USA) and imaged with an Olympus FV1000 confocal microscope utilizing Olympus FV10-ASW3.1 software.

TUNEL analysis was performed using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Millipore, Temecula, CA, USA) according to the manufacturer’s specifications. Briefly, kidney sections from each rat group were fixed using paraformaldehyde and ethanol–acetic acid solution (2:1). Sections were incubated with terminal deoxynucleotidyl transferase followed by incubation with anti-digoxigenin conjugate. Propidium iodide (1 µg/mL) was added as a nuclear counterstain. Coverslips were applied using Vectashield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA, USA). Images were obtained using confocal microscopy (LSM 510, Carl Zeiss) with 100× magnification. Whole kidney section was scanned for positive green fluorescent cells as an indicator of apoptosis (n = 4/group).