Alexa fluor 488 conjugated goat igg

Cells were grown on dishes and fixed with 4% paraformaldehyde/PBS for 10 min at room temperature and washed three times with PBS. Fixed cells were permeabilized in PBS/0.5% Triton X100 for 10 min, blocked with blocking solution (2% BSA, 2% goat serum, and 0.1% Tween20 in PBS) and incubated with a primary antibody diluted in blocking solution overnight at 4° C. The cells were then washed three times with PBS/0.01% Tween20, incubated with secondary antibodies for 1 h at room temperature, washed in PBS/0.05% Tween20, counterstained with DAPI, mounted with Vectashield (Vector Laboratories) and imaged. The following primary antibodies were used: rabbit polyclonal H3K27me3 diluted 1:5000 (Abcam), mouse SSEA-4 1:10 (DSHB), rabbit polyclonal OCT4 1:100 (Abcam), and mouse monoclonal TRA-1-60 1:500 (Abcam). The secondary antibodies used were as follows: goat anti-rabbit Alexa Fluor 488-conjugated goat IgG (Invitrogen, 1:1000) and goat anti-mouse Alexa Fluor 555-conjugated goat IgG (Invitrogen, 1:1000).

Anesthetized mice were perfused with PBS followed by 4% paraformaldehyde fixative in phosphate buffer through the left cardiac ventricle. Brains were removed after perfusion, post-fixed in 10% phosphate-buffered formalin, and processed for paraffin embedding. Brain sections of 3 μm thickness were deparaffinized and blocked in PBS containing 2% BSA and 0.5% Triton X-100 for 1 h at room temperature. The sections were then incubated with an anti-TDP-43 antibody (60019-2-Ig mouse monoclonal, Proteintech, 1:500), or an anti-Iba-1 antibody (019-19741 rabbit polyclonal, Wako, 1:1,000) at 4°C overnight, followed by an Alexa Fluor 488-conjugated goat IgG (Invitrogen, 1:1,000) for 1 h at room temperature. The sections were mounted with VECTASHIELD HardSet antifade mounting medium (Vector Laboratories) and analyzed using a fluorescence microscope (BZ-X800; Keyence). The formation of cytoplasmic inclusions and the nuclear localization of hTDP-43 in the cerebral cortex were quantitatively analyzed (∼300 neuronal cells per image, a total of ∼900 cells per mouse).