Alexa fluor 555 igg

Age-matched WT and Lsd1 KI mice were perfused with 4% paraformaldehyde (PFA) in PBS. The brains were removed and kept in 4% PFA overnight at 4 °C. Brain sections (40 μm thickness) were collected by sectioning with a cryostat and incubated in a blocking solution (2% goat serum, 0.2% Triton X-100 in PBS) for 1 h and then with the blocking solution containing an LSD1 antibody (1:500, abcam) overnight at 4 °C. Sections were then incubated with the blocking solution containing anti-rabbit Alexa Fluor 555 IgG (1:400, Invitrogen) for 2 h at room temperature. Fluorescent images were captured using a fluorescent microscope (IX51, Olympus).

Immunofluorescence was performed as described in previous studies [26 (link)]. Cells were incubated with SOX2 (AB5603 Millipore, Burlington, MA, USA) and phospho-histone3 (pH3) (ab14955 Abcam, Cambridge, UK) antibodies. Secondary antibodies of anti-mouse Alexa Fluor 555 IgG (Invitrogen, Carlsbad, CA, USA) and anti-rabbit Alexa Fluor 488 IgG (Invitrogen, Carlsbad, CA, USA) were used. Nuclear DNA was stained with Hoechst 33342 (Sigma, St. Louis, MO, USA). Pictures were taken with an Eclipse 80i microscope and processed with the NIS Elements Advances Research software (Nikon, Tokyo, Japan).

Immunofluorescence was performed following standard procedures [44 (link)]. The primary and secondary antibodies used were anti-phospho-histone H3 (PH3, 1:2000, Ab14955, Abcam, Cambridge, UK), cleaved-caspase-3 (cC3, 1:500, AF835, R&D Systems, Minneapolis, MN, USA), anti-mouse Alexa Fluor 555 IgG (1:500, Invitrogen, Carlsbad, CA, USA), anti-rabbit Alexa Fluor 488 (1:500, Invitrogen, Carlsbad, CA, USA), and anti-rabbit Alexa Fluor 555 (1:500, Invitrogen, Carlsbad, CA, USA). Nuclear DNA staining and the mounting were performed with the Vectashield Hard set Mounting Medium with DAPI counterstain (Vector Laboratories, Burlingame, CA, USA). Pictures were taken in an Eclipse 80i microscope and processed with the NIS Elements Advanced Research Software (Nikon, Tokyo, Japan).

The immunofluorescent staining of cell nuclei, EndoG, mitochondria, and γH2AX foci was performed according the following protocol: cells grown on cover slips were washed in PBS and fixed in 4% paraformaldehyde (in 1xPBS, pH 7.4) for 15 min at room temperature. After washing with PBS, cells were permeabilized using 0.1% Triton-X 100 (in 1xPBS, pH 7.4) at room temperature and then incubated with the blocking reagent (5% Bovine serum albumin in 1xPBS, pH 7.4) for 45 min. The primary antibody: EndoG (ab9647, Abcam), mitochondria antibody [MTC02] (ab3298, Abcam), and γH2AX (ab26350, Abcam) was diluted in 1% bovine serum albumin, (in1xPBS pH 7.4) and incubated with the cells at room temperature. After the incubation, cells were washed with PBS and the fluorescent-labelled secondary antibody diluted in the same buffer was added to cells (IgG-Alexa Fluor 555, A-31572 Invitrogen; IgG-Alexa Fluor 488, #4408, Cell signalling). The cells were incubated for 1 hour in the dark at room temperature. After washing, the DNA was stained with 49-6-diamidine-2-phenyl indole (DAPI, Invitrogen) diluted to a final concentration 1μg/ml in the same buffer. Cells were then washed in PBS and mounted with the anti-fade medium (Vectashield).