Astrocyte Immunostaining Protocol

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).

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Chen X., Liang H., Xi Z., Yang Y., Shan H., Wang B., Zhong Z., Xu C., Yang G.Y., Sun Q., Sun Y, & Bian L. (2020). BM-MSC Transplantation Alleviates Intracerebral Hemorrhage-Induced Brain Injury, Promotes Astrocytes Vimentin Expression, and Enhances Astrocytes Antioxidation via the Cx43/Nrf2/HO-1 Axis. Frontiers in Cell and Developmental Biology, 8, 302.

Publication 2020

rabbit anti-Nrf2 polyclonal antibody rabbit anti-HO-1 polyclonal antibody mouse anti-Cx43 monoclonal antibody rabbit anti-GFAP polyclonal antibody mouse anti-VIM monoclonal antibody 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 DAPI confocal laser-scanning microscope

Alexa fluor 488 Alexa fluor 555 Alexa fluor 647 Anti antibody Anti gfap antibody Anti igg Antibodies Astrocytes Brain Confocal laser scanning microscope Cryosections Cx43 Dapi Donkey Fluorescence Goat Monoclonal antibody Mouse Nrf2 Nuclei Rabbit

Corresponding Organization :

Other organizations : Ruijin Hospital, Shanghai Jiao Tong University, Shanghai Ninth People's Hospital, Guangdong General Hospital, Guangdong Academy of Medical Sciences

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Variable analysis

independent variables

Brain cryosections and astrocytes coverslips

dependent variables

Nrf2 expression
HO-1 expression
Cx43 expression
GFAP expression
VIM expression

control variables

Experimental procedures followed the previously described protocol (Song et al., 2019)
Primary antibody concentrations: rabbit anti-Nrf2 (1:200), rabbit anti-HO-1 (1:300), mouse anti-Cx43 (1:200), rabbit anti-GFAP (1:1000), mouse anti-VIM (1:500)
Secondary antibody concentrations (1:500)
DAPI nuclei staining (1:5000)

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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