Alexa fluor dye conjugated secondary antibody

Tissue cultures were fixed in a solution of 4% (w/v) paraformaldehyde (PFA) in PBS (0.1 M, pH 7.4) and 4% (w/v) sucrose for 1 h, followed by 2% PFA and 30% sucrose in PBS (0.1 M, pH 7.4) overnight. Cryostat sections (30 µm; 3050CM S, Leica) of fixed slice cultures were prepared and stained with antibodies against Iba1 (rabbit, 1:1,000; Fujifilm Wako #019-19741) and GFAP (mouse, 1:1,000; Sigma-Aldrich #G3893). Slices were incubated in appropriate Alexa Fluor^® dye-conjugated secondary antibodies (donkey, 1:1,000; invitrogen #A-21206). For post-hoc staining, slices were incubated with Alexa Fluor^® 488-conjugated streptavidin (1:1,000; invitrogen #S32354). TO-PRO^®-3 (1:5,000 in PBS for 10 min; invitrogen #T3605) nuclear stain or DAPI nuclear stain (1:5,000 in PBS for 20 min; Thermo Scientific #62248) was used to visualize cytoarchitecture. Sections were washed, transferred on glass slides and mounted for visualization with anti-fading mounting medium (Agilent #S302380-2). Confocal images were acquired using a Nikon Eclipse C1si laser-scanning microscope equipped with a 20x (NA 0.75; Nikon) and a 40x oil-immersion objective (NA 1.3; Nikon).

Cells were fixed with 4% paraformaldehyde and 4% sucrose in PBS at room temperature for 30 min. Fixed cells were then permeabilized with 1% SDS for 5 min, blocked in 5% bovine serum albumin (Sigma) in 0.1% Triton X-100 (Sigma) in PBS (PBST) for 2 h, and incubated with rabbit anti-AC3 (1:400; Santa Cruz Biotechnology) and TUJ-1 (1:1000; Promega) at 4°C overnight. After washing 3 times in PBST, cells were incubated with appropriate Alexa Fluor dye-conjugated secondary antibodies (Invitrogen) for 2 h. Cells were finally counterstained with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, 2 μg/mL; Invitrogen) and mounted onto slides using Aqua-Poly/Mount (Polysciences).

The cultured cells were fixed in 4% paraformaldehyde (Santa Cruz Biotechnology, Dallas, TX, USA, cat# sc-281692) at room temperature for 10 min and then washed with D-PBS (Corning, Corning, NY, USA, cat# 21-030-CV). The cells were incubated overnight with primary antibodies (Supplementary Table 1) at 4°C following incubation in blocking buffer (4.5% cold fish gelatin, 0.1% sodium azide, 5% BSA, and 0.25% Triton X-100 in PBS) for 1 h at room temperature. The samples were stained with Alexa Fluor Dye-conjugated secondary antibodies (1:1,000; Invitrogen) and then counterstained with Hoechst 33342 (1:1,000; Thermo Fisher Scientific). The fluorochrome-labeled proteins were detected using Leica SP5-II confocal microscope (Leica Microsystems) with a 100 × objective and a fluorescence microscope (Axioplan; Carl Zeiss Meditec GmbH, Oberkochen, Germany) with a 40 × objective.

To perform immunocytochemical analysis, cells were fixed with 4%PFA (pH = 7.4) for 30 min at room temperature. Fixed cells were washed three times with PBS and were incubated in 1% TritonX‐100 for 30 min, followed by blocking with 2% BSA for 1 h. Then, immunostaining was performed according to standard protocols using the following primary antibodies: OCT3/4, NANOG, KLF4, SP5, GATA3, KRT7, Brachyury, PAX6, and SOX17. Appropriate Alexa Fluor dye‐conjugated secondary antibodies (Invitrogen) were used. Nuclei were stained with DAPI (Life Technologies). Images were taken with a confocal microscope (Zeiss). Data analysis were performed with ImageJ.

For immuno-staining of Dazl protein, cells were fixed with 4% paraformaldehyde in PBS for 1 hr at room temperature, permeabilized with 0.3% Triton-X in PBS for 15 min. For immuno-staining of Vasa protein, cells were fixed with 4% paraformaldehyde in PBS for 1 hr at room temperature, incubated with 0.2 M Glycine for 10 min at room temperature. Then the cells were re-fixed with 100% methanols for 10 min at −20 °C, and were incubated with blocking solution (10% FBS, 1% BSA, 0.1% Triton-X in PBS) for 1 hr at 4 °C. The cells were then incubated with primary antibody (rabbit anti-Dazl, Abcam; rabbit anti-Vasa49 ) in the blocking solution overnight at 4 °C. The cells were then washed and incubated with secondary antibody (Alexa Fluor dye-conjugated secondary antibodies, Invitrogen) and 3 μg/ml⁻¹ 4′,6-diamidino-2-phenylindole (DAPI) in blocking solution for 1 hr at room temperature. The cells were washed again and mounted with VECTASHIELD (Vectro Laboratories). Stained cells were observed under a Leica AF6000 fluorescence microscope, and were then analyzed with image J. The immune-staining of Sycp3 protein were performed as described previously using anti-Sycp3 (Abcam)40 .

Human recombinant BDNF was purchased from PeproTech (Rocky Hill, NJ, United States). Rabbit anti-Akt and rabbit anti-phospho-AktS473 were obtained from Cell Signaling Technology (Danvers, MA, United States). Chicken anti-microtubule-associated protein 2 (MAP2) and rabbit anti-hemagglutinin (HA) were obtained from Abcam (Cambridge, MA, United States). Alexa Fluor® dye-conjugated secondary antibodies were obtained from Invitrogen (Carlsbad, CA, United States). Horseradish peroxidase (HRP)-conjugated goat anti-mouse or rabbit IgG and HRP-conjugated rabbit anti-goat IgG were purchased from Calbiochem (La Jolla, CA, United States). Recombinant anti-CREB (phospho S133) antibody [E113] (ab32096), anti-CREB antibody (ab31387), and mouse anti-GFP were obtained from Abcam (Boston, MA, United States). Ki16425 was purchased from Sigma-Aldrich. All cell culture reagents were purchased from Invitrogen and all the other reagents were purchased from Sigma-Aldrich.