Leica dfc350 fx camera

Cells were fixed in 2% formaldehyde and permeabilized in 0.25% Triton X-100 in PBS for 5 min at room temperature. For immunolabeling, cells were incubated with anti-PML (goat polyclonal N19 Santa Cruz Biotechnology) and anti-TRF1 (rabbit polyclonal N19, Santa Cruz Biotechnology, Dallas, TX, USA) primary antibodies at room temperature for 2 h, then washed in PBS and incubated with the FITC-conjugated bovine anti-goat (Santa Cruz Biotechnology, Dallas, TX, USA) followed by cy3-conjugated donkey anti-rabbit secondary antibodies (Jackson Immunoresearch). For FISH analysis, cells fixed as above were dehydrated and hybridized with Cy3-coupled PNA telo probe (Panagene) as described by Lenain et al. (2006). Images were captured with an X100 objective. Fluorescent signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by a Leica FW4000 deconvolution software (Leica, Solms, Germany). This system permits to focus single planes inside the cell, generating 3D high-resolution images. For qFISH analysis, telomeric spots intensity was calculated by ImageJ software on images acquired at the same exposure time.

After perfused through the heart with PBS or 0.9% normal saline followed by 4% paraformaldehyde under sodium pentobarbital anesthesia (50 mg/kg, i.p.), the excised spinal cord was fixed in 4% paraformaldehyde for at least 30 min. Subsequently, the spinal cord segments were transferred into 30% sucrose to be fully dehydrated at 4 °C. The tissue was cut into 20-μm-thick sections. After three washes in PBS, sections were blocked with immunofluorescent blocking agent (Beyotime) for 1 h at 20–25 °C and then incubated with the following primary antibodies: BDNF (1:500; ab213323, Abcam), phospho-TrkB (Tyr816, 1:200; NBP1-03499, Novus Biological), OX-42 (1:500; ab1211, Abcam), NeuN (1:200; MAB377, Millipore), and GFAP(1:400; #3670, Cell Signaling Technology) at 4 °C overnight. The antibodies used above were all within their validity period. After three washes with PBS, the sections were incubated with the secondary antibodies conjugated with Cy3 or Alexa-488 without light for 1 h at 20–25 °C. Measuring and imaging were performed by using a Leica fluorescence microscope (Leica DFC350 FX camera). We used the same exposure, gain, and gamma every time to keep the images standardized. ImageJ was also used to quantify the fluorescent intensity of each image.

Under anesthesia, rats were perfused with 4% paraformaldehyde through the ascending aorta. The L6-S1 spinal cord section was removed and postfixed in paraformaldehyde for 30 min. The spinal cord was then transferred to 30% sucrose for dehydration at 4°C. Tissues were sectioned (25 μm thickness) and processed for immunofluorescence staining. Sections were blocked for 1 h and then incubated with primary antibodies against Iba-1 (1 : 200; Abcam), CD11b (OX-42, 1 : 400; Abcam), TNF-α (1 : 200; Bioworld), and IL-1β (1 : 500; Abcam), overnight at 4°C. After that, the sections were incubated in Cy3 or Alexa-488 conjugated secondary antibodies (Jackson Laboratories, Bar Harbor, ME, USA) for 1 h at room temperature. A Leica fluorescence microscope (Leica DFC350 FX camera; Wetzlar, Germany) was used to measure and image the stained section. To quantify Iba-1, OX-42, TNF- α, and IL-1β expression in the L6-S1 SDH, the fluorescence intensity of each area was analyzed with ImageJ software. To verify the specificity of primary antibodies, immunostaining was also performed in parallel but without primary antibodies (data not shown).