Alexa fluor 555 donkey anti rabbit igg

Lung tissues were washed in PBS and fixed in 10% PBS-buffered formalin for 24 h. Tissues were then embedded in optimal cutting temperature compound, and 5–6 μm sections were cut by Microtome Cryostat (Leica CM3050S, Leica Biosystems), and stained with antibody according to the protocol provided by the manufacturer. Cells, cultured on the microscope cover glass, were washed in PBS and fixed in 10% PBS-buffered formalin for 15 min, and then stained with antibodies according to the protocol provided by the manufacturer. The sections were examined using a laser scanning confocal microscopy (Leica Biosystems). Primary antibodies (1 : 200) were applied as follows: Rat anti-mouse C3 (Abcam ab11862), Rabbit anti-C3 (Abcam ab200999), Rat anti-mouse Nestin (Abcam ab81462), Rat anti-mouse Ly6G (Abcam ab25377), Rabbit anti-human C3 (Abcam ab97462), Mouse monoclonal anti-human Nestin (Abcam ab22035), Rabbit anti-Histone H3-cit (Abcam ab5103), and Rabbit anti-Myeloperoxidase (Abcam ab208670). The following secondary antibodies (1 : 1000) were used: Alexa Fluor 488 Goat anti-Rat IgG (Abcam ab150157), Alexa Fluor 488 Goat anti-Rabbit IgG (Abcam ab150077), Alexa Fluor 555 Donkey anti-Rabbit IgG (Beyotime A0453), Alexa Fluor 647 Goat anti-mouse IgG (Beyotime A0473), and Alexa Fluor 594 Goat anti-Rat IgG (Abcam ab150160).

Brain cryosections and astrocytes coverslips were immunostained with following primary antibodies: rabbit anti-YAP polyclonal antibody (1:100, Santa Cruz Biotechnology, USA), rabbit anti-Ki67 polyclonal antibody (1:500, Abcam), rabbit anti-GFAP polyclonal antibody (1:1000, Servicebio, China), mouse anti-VIM monoclonal antibody (1:500, Servicebio), mouse anti-GFAP monoclonal antibody (1:500, Servicebio). Secondary antibodies used included Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 555 donkey anti-rabbit IgG, Alexa Fluor 555 donkey anti-mouse IgG and, and Alexa Flour 647 goat anti-mouse IgG (1:500, Beyotime). Nuclei were stained with DAPI (1:3000, Beyotime). The fluorescence images were observed and analyzed by confocal laser-scanning microscope (Leica).

Cells were seeded on coverslips in 24‐well plates and fixed with 4% paraformaldehyde. After 15 mins, cells were permeabilized with phosphate‐buffered saline (PBS) containing 0.2% Triton X‐100 (PBST) for 5 min and then blocked with 1% bovine serum albumin in PBST. Immunostaining was performed using primary antibodies, rabbit anti‐cleaved‐Notch1 (Immunoway, plano, TX, USA) and mouse anti‐MCM5 (Proteintech, Chicago, IL, USA), rabbit anti‐Keratin 5 (Abcam, Cambridge, UK) and mouse anti‐Vimentin (Huaan Biotechnology, Hangzhou, China), respectively overnight at 4 °C. After PBS washing for three times, the slides were incubated with Alexa Fluor 555 donkey anti‐rabbit IgG and Alexa Fluor 488 goat anti‐mouse IgG (Beyotime, Beijing, China) at room temperature for 1 h. The slides were counterstained with DAPI (Beyotime, Beijing, China) and images were captured using the Zeiss microscope (Carl Zeiss, Jena, Germany).

Brain cryosections and astrocytes coverslips were performed as previously described (Song et al., 2019 (link)), and immunostained with following primary antibodies: rabbit anti-Nrf2 polyclonal antibody (1:200, Santa Cruz Biotechnology, United States), rabbit anti-HO-1 polyclonal antibody (1:300, Abcam, United Kingdom), mouse anti-Cx43 monoclonal antibody (1:200, Invitrogen, United States), rabbit anti-GFAP polyclonal antibody (1:1000, Servicebio, China), mouse anti-VIM monoclonal antibody (1:500, Servicebio). The secondary antibodies used (1:500, Beyotime): Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 555 donkey anti-mouse IgG, Alexa Fluor 555 donkey anti-rabbit IgG, Alexa Fluor 647 goat anti-mouse IgG, Nuclei were stained with DAPI (1:5000, Beyotime). The fluorescence images were observed and analyzed by a confocal laser-scanning microscope (Leica).