Alexa 488 conjugated goat anti rabbit igg h l

Cell surface binding assays were performed essentially as described^{27 (link), 29 (link)}. COS-7 cells were transfected with the expression vectors for candidate binding proteins and maintained for 24 h. The transfected COS-7 cells were washed with extracellular solution (ECS, containing 168 mM NaCl, 2.4 mM KCl, 20 mM HEPES (pH 7.4), 10 mM d-glucose, 2 mM CaCl₂ and 1.3 mM MgCl₂) that contained 100 μg ml^–1 bovine serum albumin (BSA) and then incubated with ECS and BSA containing 100 nM purified Fc-fusion protein for 1 h at room temperature. These cells were washed in ECS, fixed with parafix solution, incubated with blocking solution followed by rabbit anti-HA without permeabilization and then immunostained with Alexa488-conjugated goat anti-rabbit IgG (H+L) (1:500; Invitrogen) and Alexa594-conjugated donkey anti-human IgG (H+L) (1:500; Jackson ImmunoResearch) to label surface HA and bound Fc proteins, respectively. Images were acquired on a Leica DM6000 fluorescent microscope with a 40 × 0.75 NA dry objective and a Hamamatsu cooled CCD camera using Volocity software (Perkin Elmer).

In this study, we used rabbit anti‐human HMGB1, anti‐human p62, anti‐human Nrf2 (Abcam, Shanghai, China), anti‐human LC3, anti‐human Keap1 (Cell Signaling Technology, Danvers, MA, USA), and Alexa488‐conjugated goat anti‐rabbit IgG (H+L) (Invitrogen, Carlsbad, CA, USA) antibodies. Analytical pure grade H₂O₂ was purchased from TianJin Chemical Reagent Factory (Tianjin, China). Propidium iodide (PI) was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Recombinant human HMGB1 (rHMGB1) was purchased from R&D Systems Inc. (Minneapolis, MN, USA).

The fixation and permeabilization of antheridia were carried out essentially according to previous report⁵³ . Isolated spermatid cells were incubated with a polyclonal anti-centrin antibody against a recombinant protein corresponding to a full-length centrin from a brown alga Scytosiphon lomentaria (a gift from Dr. Taizo Motomura, Hokkaido University, Japan) diluted 1:500 with PBS [137 mM NaCl, 2.68 mM KCl, 8.1 mM Na₂HPO₄, and 1.47 mM KH₂PO₄, pH 7.4] in a moist chamber for 90 min at 37 °C. After a PBS wash, incubation with a monoclonal anti α-tubulin (T5168, Sigma) diluted 1:500 with PBS was performed for 60 min at 37 °C. After a wash again with PBS, the samples were incubated for 60 min at 37 °C with an equal mixture of Alexa 488-conjugated goat anti-rabbit IgG (H + L) (A11034, Invitrogen) diluted 1:200 with PBS and Alexa 568-conjugated goat anti-mouse IgG (H + L) (A11031, Invitrogen) diluted 1:200 with PBS. After washing with PBS for 10 min, the nuclei were stained with 1 μg ml⁻¹ DAPI in PBS. Slides were mounted using Vectashield (Vector Laboratory) and observed using a confocal laser scanning microscope (FV-1000, Olympus).