Tsc sp5 confocal microscope

Gastrocnemius muscles from C57BL/6J mice were collected for the whole mounting stain.⁵ Briefly, the muscle was fixed with 2% paraformaldehyde for 2 h at room temperature, embedded in optimum cutting temperature (OCT) compound, and sectioned to 20 μm thickness. Sections were incubated with anti‐NRIP (GeneTex, Irvine, CA, USA; 1:200), anti‐rapsyn (Abcam, Cambridge, MA, USA; 1:100) and anti‐ACTN2 (Abcam; 1:500) primary antibodies overnight at 4°C and then incubated with a mixture containing 488‐conjugated donkey anti‐rabbit secondary antibody and Alexa‐594‐conjugated α‐bungarotoxin (α‐BTX) and mounted in DAPI Fluoromount‐G (SouthernBiotech, Birmingham, AL, USA). The fluorescent images were visualized by Leica TSC SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).

NTera2/D1 cells were washed with PBS, fixed with ice-cold methanol for 15 min, and incubated with γH2A.X- or 53BP1-specific primary antibody for 1 h. Alternatively, they were washed with PBS, fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 100 μg/ml digitonin (Life Technologies) in PBS for 15 min, and incubated with LC3B-specific primary antibody for 1 h. Cells were then washed three times with PBS, incubated for 30 min with appropriate Alexa Fluor 488-conjugated or Alexa Fluor 555-conjugated secondary antibodies and washed again three times in PBS. Nuclei were stained with 1.5 μM 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich) in PBS for 5 min. Coverslips were mounted in Fluorescence Mounting Medium (Dako, Milan, Italy). Samples were visualized on a TSC SP5 confocal microscope (Leica Microsystems, Milan, Italy) installed on an inverted LEICA DMI 6000CS microscope, using PlanApo 40× 1.25 NA objective or PlanApo 63× 1.4 NA oil immersion objectives. Images were acquired using the LAS AF acquisition software (Leica Microsystems). Fluorescence intensity measurements were performed using the Quantitation Module of Volocity software (Perkin Elmer Life Science, Milan, Italy). At least five representative fields were acquired and analyzed for each sample.

Eyeballs were dissected and fixed in 4% paraformaldehyde (Cat. No. BM-698; Bostonbioproducts, BioProducts, Ashland, MA) for 2 hours at room temperature followed by immersing into 20% sucrose solution in phosphate buffered saline (PBS) for 2 hours. The eyeballs were embedded in O.C.T. compound (Cat. No. 4583; Sakura Finetek USA, Inc., Torrance, CA) on dry ice. Frozen sections of the retina (10 μm) were incubated with a blocking buffer containing 4% bovine serum albumin and 0.5% Triton X-100 in PBS for 1 hour followed by incubation with a primary antibody against β-III-tubulin (1:500; Cat. No. MAB5564, Millipore), Recoverin (1:500; Cat. No. AB5585, Millipore) or H3K27me3 (1:500; Cat. No. 9733S, Cell Signaling Technology, Danvers, MA) in the blocking buffer for overnight at 4°C. Slides were washed with PBS 3x at 10 min. each before incubation with Cy3/Cy2-conjugated secondary antibody in the blocking buffer [Cy3-AffiniPure Donkey Anti-Mouse (1:500; Cat. No. 715-165-151, Jackson ImmunoResearch) or Cy2-AffiniPure Donkey Anti-Rabbit (1:500; Cat. No. 711-095-152, Jackson ImmunoResearch)] were applied for 1 hour at room temperature. Slides were washed with PBS 3 x at 10 min. each. The slides were mounted in a mounting media containing DAPI (Cat. No. H-1200; Vector Laboratories Inc.) and imaged with a TSC SP5 confocal microscope (Leica Microsystems, Richmond, IL).

The procedure for tissue preparation and staining was as previously reported^{50 (link)}. Briefly, fixed specimens were embedded in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan) and sectioned at 20 or 50 μm. For immunohistochemistry, the following antibodies were used: Armenian hamster anti-mouse CD31 mAb (MAB1398Z; Merck Millipore, Darmstadt, Germany; dilution 1/200), rabbit anti–claudin-5 pAb (polyclonal antibody; Abcam, Cambridge, MA, USA; dilution 1/200), rabbit anti-neural/glial antigen-2 (NG2) pAb (AB5320, Sigma–Aldrich, St. Louis, Missouri, USA; dilution 1/250), rabbit anti-glial fibrillary acidic protein (GFAP) pAb (HPA056030; Sigma–Aldrich; dilution 1/1000), Alexa Fluor 546–conjugated goat anti-rabbit IgG pAb (A-11010; Thermo Fisher Scientific; dilution 1/200), and Alexa Fluor 647–conjugated goat anti-Armenian hamster IgG pAb (127-605-160; Jackson ImmunoResearch Laboratories, West Grove, PA, USA; dilution 1/400). The sections were visualized using a Leica TSC SP5 confocal microscope and processed with Leica Application Suite (Leica Microsystems, Wetzlar, Germany) and Adobe Photoshop CC software (Adobe Systems, Mountain View, CA, USA).

For immunofluorescence experiments, A375 cells were seeded at a density of 5 × 10^{3 (link)} in 12-well cluster plates in DMEM supplemented with 1% FBS. After 24 h, cells were treated with compounds 1 or 2 (0.5 and 1 μM for 48 h). Cells were then washed with phosphate-buffered saline (PBS), fixed with ice-cold methanol for 5 min and permeabilized with Triton 0.2% in PBS for 10 min. Immunostaining of γH2AX was performed as previously described^{57 (link)}. For Wheat Germ Agglutinin (WGA) immunostaining, cells were labeled as previously described^{58 (link)}. Samples were visualized on a TSC SP5 confocal microscope (Leica Microsystems, Milan, Italy) installed on an inverted LEICA DMI 6000CS microscope, using PlanApo 40 × 1.25 NA objective or PlanApo 63 × 1.4 NA oil immersion objectives. Images were acquired using the LAS AF acquisition software (Leica Microsystems). Fluorescence intensity measurements were performed using the Quantitation Module of Volocity software (Perkin Elmer Life Science, Milan, Italy).