Lsm 700 confocal laser scanning microscope

We seeded 5 × 10⁴ RAW 264.7 cells/mL in a confocal chamber slide and pre-treated them with 10 μg/mL IPA for 2 h. Subsequently, we treated the cells with 100 ng/mL RANKL in the presence of IPA for 9 h and 5 min. IF staining was performed as described by Cho et al. with some modification [35 (link)]. The cells were incubated overnight at 4 °C with the following primary antibodies: anti-NFATc1 (1:100), anti-c-Fos (1:100), and anti-p65 (1:100). Subsequently, the cells were incubated for 1.5 h with the following secondary antibodies at room temperature: Alexa fluor488-labeled goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody and Alexa fluor488-labeled goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody (1:800; Thermo Fisher Scientific, Inc., Foster City, CA, USA). Nuclei were stained with 40 μg/mL Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Eventually, fluorescent signals were detected using an LSM 700 Zeiss confocal laser scanning microscope (Carl Zeiss, Jena, Germany).

Lipid peroxidation in oocytes was assessed by fluorescent staining using BODIPY™ fluorescent reagent (Invitrogen, Waltham, MA, USA). Denuded oocytes were incubated in a drop of PBS-PVP with BODIPY™ (10 μM) under the mineral oil at 37 °C and 5% CO₂ for 30 min. Afterward, oocytes were washed in PBS-PVP three times and mounted on a glass slide. Stained oocytes were then scanned by an LSM 700 Zeiss confocal laser scanning microscope (Carl Zeiss Slovakia, s.r.o., Bratislava, Slovak Republic) and the lipid peroxidation level was evaluated using ImageJ software.

Following electrophysiological recordings coronal or parahorizontal brain slices were fixed in 4% paraformaldehyde in 0.12 M phosphate buffer at pH 7.4 overnight. Slices were then washed in PBS and mounted with Vectashield® mounting medium on plus polarized glass slides (Super Frost Plus Thermo Scientific) and coverslipped. All images were acquired with a LSM700 Zeiss confocal laser scanning microscope (Zeiss, Göttingen, Germany). Fluorescent signals were recorded using 488 nm as wavelength for excitation. To obtain images of slices derived from Th1ChR2-EYFPmice we used low magnification objectives, 1.25× and 5×, with an additional digital zoom factor of 0.5. The confocal pinhole was kept at 1, the gain and the offset were adjusted to prevent saturation of the signal. Single section confocal images (1024 × 1024) were collected and exported in TIFF file format. Background subtraction was performed on all images by means of ImageJ software.

The level of intracellular ROS was assessed by fluorescent staining using CellROX Green (Invitrogen, MA, USA). Denuded oocytes were incubated for 30 min in a dye mixture (according to the producer´s guide), washed in PBS–PVP, fixed for 10 min in 4% of neutrally buffered formalin, covered with a drop of Vectashield anti-fade mounting medium with DAPI, and mounted onto glass slides with a coverslip. Stained oocytes were scanned by an LSM 700 Zeiss confocal laser scanning microscope, and the ROS formation was evaluated based on fluorescence intensity using ImageJ software and the formula described above.

Deeply anesthetized mice were perfused with 4% paraformaldehyde in 0.12 M sodium phosphate buffer (pH 7.5). Brains were postfixed overnight in the same solution and stored at 4°C. Thirty‐micrometer sections were cut using a vibratome (SM 2010R, Leica, Milan, Italy) and maintained in 30% sucrose overnight at 4°C. Then sections were dehydrated with serial alcohol dilutions (50–70–50%) and incubated 1 hour at room temperature in a solution containing 10% donkey serum in phosphate‐buffered saline (PBS) 0.25%‐Triton X‐100 (PBS‐Tx). Finally, they were incubated 3 days at 4°C with the following primary antibodies: goat anti‐ChAT (choline acetyltransferase) (1:500, NBPI30052, Novus Biologicals, Bio‐Techne, Milan, Italy) and rabbit anti‐VAChT (1:500, 139103, Synaptic Systems, Goettingen, Germany). Sections were rinsed thrice for 10 minutes in Tris Buffered Saline (TBS) and incubated at room temperature for 2 hours with 1:200 Alexa 488 or Alexa 647 (Invitrogen, Thermo Fisher Scientific, Milan, Italy). Sections were rinsed for 10 minutes thrice in TBS before mounting with Super Frost Plus (Thermo Scientific, Rodano (MI) Italy). Images were acquired using an LSM700 Zeiss confocal laser scanning microscope (Zeiss, Milan, Italy); confocal images were acquired as previously described.³¹, ³³

Mice were perfused transcardially with 4% paraformaldehyde after being euthanized with pentobarbital. Brain tissue was dehydrated in 10% sucrose overnight and embedded in a solution with 12% gelatin and 10% sucrose. Sagittal sections of 40 μm were cut using a microtome (SM2000R; Leica Microsystems). Primary antibody staining was performed using anti-UBE3A (mouse E6AP clone 3E5, MilliporeSigma, SAB1404508) at a concentration of 1:750 and anti-parvalbumin (rabbit, Swant, PV27) at a concentration of 1:5000. Secondary antibody staining was performed using donkey anti-mouse 488 (Jackson ImmunoResearch, 715545150, 1:200) and donkey anti-rabbit Cy3 (Jackson ImmunoResearch, 711165152, 1:200), and it was followed by DAPI staining for 10 minutes. Tissue slices were mounted on 24 × 40 mm coverslips. Images were taken using LSM700 Zeiss Confocal Laser Scanning Microscope and analyzed using Fiji.

The intracellular ROS was assessed by fluorescent staining using fluorescent reagent CellROX™ Green (Invitrogen, Waltham, MA, USA). Denuded oocytes were incubated in a dye mixture (according to the manufacturer) for 30 min at 37 °C, washed in PBS-PVP solution (phosphate-buffered saline with 0.6% of polyvinylpyrrolidone), fixed in 4% formalin for 10 min, and mounted on a glass slide. Stained oocytes were scanned by an LSM 700 Zeiss confocal laser scanning microscope, and the ROS formation level was evaluated using ImageJ 1.53a software.