Alexa fluor 488 anti rat

The fresh brain tissue was fixed for overnight in 4% PFA in PBS at 4 °C, rinsed in PBS and dehydrated in 30% sucrose in PBS at 4 °C. Dehydrated tissue embedded and frozen at −80 °C in optimum cutting temperature compound (OCT; Tissue-Tek) and sectioned at 30μm using cryostat microtome (Leica CM1950). Slices were washed with PBS, blocked with 5% BSA, 1% Normal Goat Serum (Jackson ImmunoResearch, 005000121) and 0.3% Triton X-100 PBS for 1 h at room temperature, followed by incubation with the primary antibodies overnight at 4 °C. The primary antibodies used were anti-SST (rat, Millipore, MAB354, 1:50), anti-NOS1 (rabbit, CST, 4231S, 1:200), NR2F2 (mouse, Abcam, ab41859, 1:200). Sections were washed three times in PBS and incubated with secondary antibodies at room temperature for 1 h. The secondary antibodies used were Alexa Fluor 488 anti-Rat (Invitrogen, A11006, 1:1000), Alexa Fluor 594 anti-Rabbit (Invitrogen, A11037, 1:1000), and Alexa Fluor 594 anti-Mouse (Invitrogen, A-11032, 1:1000). Sections were washed three times in PBS and stained with DAPI (Abcam, ab104139). Slides were imaged with a confocal microscope (Zeiss LSM 880) and analyzed using Zeiss ZEN software suites (blue edition). Images were acquired with 1 AU pinhole size using 20x or 63x objectives at the resolution of 1024×1024 pixels. Stacks of optical slices were collected through the entire z-axis of each slice.

Cells were labeled with 100 µM iododeoxyuridine (IdU) for 10 min and then labeled with100 µM chlorodeoxyuridine (CldU) for 20 min for mock experiment. For monitoring DNA synthesis during replication stress, cells were labeled with IdU (100 µM) for 10 min, followed by exposure to CldU (100 µM) coupled with hydroxyurea (5 mM for HCT115 and 0.5 mM for human fibroblasts) for 1 h. DNA fibers were spread as described previously (29 (link)) and stained with primary antibodies (mouse anti-BrdU/IdU from BD Bioscience; rat anti-BrdU/CldU from Accurate Chemical) and fluorescence-conjugated secondary antibodies (Alexa Fluor 488-anti-rat and Texas Red-conjugated anti-mouse from Invitrogen). Fibers were imaged using Zeiss Axio Imager M2 and measured using AxioVision software (x64 version 4.8.3.0).

Mice were anaesthetized with 200 mg/mL sodium pentobarbital and intracardially perfused with PBS, pH 7.4, and 4% paraformaldehyde. The brains were post-fixed and cut into six series of coronal sections (40 µm) using a vibratome (Leica VT1000S, Leica Biosystems). The following primary antibodies were used to identify microglia and immature neurons: rabbit anti-Iba1 (1:500; Wako, ref: 019-19741) and goat anti-DCX (1:200; Santa Cruz Biotechnology, ref: sc-8066). Then, we used the corresponding biotinylated secondary antibodies: anti-rabbit (1:1000; Dako, ref: E0432) and anti-goat (1:1000; Dako, ref: E0466) [36 (link)].
To identify new neurons, control and stress mice received three intraperitoneal administrations of BrdU (50 mg/kg, Sigma–Aldrich, Madrid, Spain) separated by 3 h just after acute stress treatment. Double immunofluorescence labelling was carried out combining the rat anti-BrdU antibody (1:1000; Accurate Chemical, ref: OBT0030) and the rabbit anti-DCX antibody (1:600; Abcam, ref: ab18723), followed by the corresponding secondary antibodies of Alexa Fluor 488 anti-rat (1:1000; Invitrogen, ref: A21208) and Alexa Fluor 568 anti-rabbit (1:1000; Invitrogen, ref: A10042). DAPI was used as a nuclear contrast stain [36 (link),37 (link)] (see Supplementary Information for details).