Alexa fluor 488 conjugated goat anti mouse igg h l

Double immunofluorescence staining experiments were performed as per methods previously described by our laboratory (22 (link)). Before immunofluorescence staining, frozen temporal cortex tissue sections (6-µm) were warmed at 26˚C for 30 min and fixed in ice-cold acetone or other alternate 4% paraformaldehyde fixatives for 10 min. The sections were blocked in 5% FBS for 60 min and incubated with primary antibodies (Anti-NeuN antibody-Neuronal Marker; 1:100; cat. no. ab177487; Abcam; Anti-GFAP antibody; 1:100; cat. no. ab7260; Abcam; and Cdk5-antibody; 1:50; cat no sc-6247; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The sections were washed and incubated with appropriate secondary antibodies (Alexa Fluor^® 594-conjugated goat anti-rabbit IgG H&L; 1:300, cat. no. ab150080; Abcam; and Alexa Fluor^® 488-conjugated goat anti-mouse IgG H&L; 1:300, cat. no. ab150113; Abcam) for 1 h at 26˚C. Cell nuclei were stained using 4-diamidino-2-phenylindole (DAPI). Fluorescence microscopy was performed with a ZEISS HB050 inverted microscope (magnification, x40; Carl Zeiss AG) and six views of fields were processed using Image-Pro Plus 7.0 (Media Cybernetics, Inc.).

The spleen leukocytes were centrifuged onto slides using Cytospin 4 (Thermo Scientific, USA) at 1 000 ×g for 3 min, then fixed with methyl alcohol for 5 min. After the slides were blocked with 1% bovine serum albumin (BSA) at 37 °C for 1 h, the samples were washed twice with PBST and once with PBS. The samples were subsequently incubated with 1:200 diluted mouse-anti CD3ε or CD28 mAbs, rabbit anti-p-S6 (Ser240/244), rabbit anti-p-ERK1/2 (Thr202/Tyr 204), or mouse anti-β-actin primary antibodies at 37 °C for 1 h. After washing, the cells were incubated with 1:800 diluted Alexa Fluor 488-conjugated goat anti-mouse IgG H+L (Abcam, UK), Alexa Fluor 594-conjugated goat anti-mouse IgG H+L (Abcam), or Alexa Fluor 594-conjugated goat anti-rabbit IgG H+L (Abcam, UK) secondary antibodies at 37 °C for 1 h. For the CD3 and CD28 co-localization assay, the cells were first stained with mouse anti-CD28 mAbs and Alexa Fluor 594-conjugated goat anti-mouse IgG H+L, followed by incubation with FITC-conjugated mouse anti-CD3ε mAbs at 37 °C for 1 h. After washing three times, 4’,6-diamidino-2-phenylindole (DAPI) staining solution (Beyotime, China) was added, with the slides then sealed with a coverslip and observed using a Zeiss ApoTome microscope (Germany).