Anti mouse igg h l alexa fluor 488

The general flow cytometry analysis protocols were performed as follows: The cells were centrifuged for 5 min at 400 x g and 10˚C. Single cell suspensions were obtained in a solution consisting of PBS supplemented with 2% FBS, and then re-suspended in 200 µl 4% paraformaldehyde and fixed at room temperature for 10 min. The cells were permeabilized using 100 µl PBS with 0.1% Triton X-100 (cat. no. ST797; Beyotime Institute of Biotechnology) for 20 min at 4˚C. The primary antibodies were added and cells were incubated at 4˚C for 30 min, and then subsequently, cells were treated with the corresponding secondary antibody at 4˚C for 30 min. Next, flow cytometry was performed using a BD Fortessa (Becton-Dickinson and Company). Analysis of the flow data was performed using FlowJo version 10 (FlowJo, LLC). The primary antibodies used were mouse anti-Nestin polyclonal antibody (1:200; Abcam; cat. no. ab1642) and mouse anti-GFAP polyclonal antibody (1:200; Abcam; cat. no. ab10062). The secondary antibody used was an anti-mouse IgG H&L-AlexaFluor 488 (1:1,000; Abcam; cat. no. ab150105).

Five μm thickness of brain sections from GPx-1 KO mice were positioned on the same slide and processed for immunostaining. Adhered tissues on poly-L-lysine-precoated coverslips, were fixed in PBS-4% para-formaldehyde (PFA), and were permeabilized with 0.1% Triton X-100 in PBS for 15 min. After saturation with PBS-1% BSA, tissues were incubated for 40 min with the primary antibody and were incubated for 40 min with the secondary antibody as follows: mouse anti-Nrf2 (1:100) (Santa Cruz, TX, USA), goat anti-ChAT (1:100) (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were anti-mouse IgG H&L (Alexa Fluor® 488) (1:200) (Abcam, Cambridge, MA, USA), anti-goat IgG H&L (Alexa Fluor® 546) (1:200) (Invitrogen, Carlsbad, CA, USA) [33 (link)]. Please refer to Supplementary Materials and Methods 1.12.