Alexa 594 donkey anti mouse igg

The antibodies used in the present study were rabbit polyclonal antibody against rainbow trout GR (1:250) [29 (link)], Caveolin-1 mouse monoclonal antibody (1:500) [30 (link),31 (link)], ORAI-1 rabbit polyclonal antibody (1:250) [22 (link),32 (link),33 (link)], and phalloidin conjugated TRITC (1:500) for F-actin detection (1:500) [34 (link)]. The secondary antibodies used were Alexa 488 goat anti-rabbit IgG and Alexa 594 donkey anti-mouse IgG (Invitrogen, Burlington, ON, Canada).

PC12 cells were seeded in a cover glass chamber (5222-004, Iwaki), and after confirming adhesion, PACAP was added to a working concentration of 10⁻⁷ M and the cells were cultured for 3 days. After removing the medium, the cells were washed with PBS and fixed with a 4% PFA solution. After washing again, the cells were treated with PBS solution containing 0.1% Triton X 100 for 10 minutes. After treatment and washing with PBS, blocking was carried out with PBS solution containing 3% bovine serum albumin (BSA) (Sigma-Aldrich) and 0.1% Tween 20, followed by incubation using a mouse anti-α-tubulin antibody (Abcam Inc., 1 : 1000) overnight at 4°C. The cells were then washed with PBS and crossreacted with Alexa 594 donkey anti-mouse IgG (Invitrogen, 1: 1000) as a secondary antibody at room temperature for 60 minutes. After washing again with PBS, Alexa Fluor 488-labeled rabbit anti-Neu-N antibody (Abcam, 1 : 50) was crossreacted overnight at 4°C. Postrinsing with PBS, nuclear-specific staining was performed with DAPI (D 1306; Thermo Fisher Scientific, 1: 10000) for 3 minutes at room temperature and the cells were finally washed with PBS and sealed. After drying, the cells were observed under a fluorescence microscope BZ-X 710 (Keyence).

Embryos were fixed with 4% paraformaldehyde in DPBS for 30 min at room temperature and then washed three times with 0.2% BSA in DPBS. Embryos were then permeabilized at room temperature in 0.5% Triton X-100 in DPBS for 30 min. After washing according to the above method, embryos were incubated with primary antibodies in 0.2% BSA for 4 h at room temperature, washed with 0.2% BSA and incubated with secondary antibodies in 0.2% BSA for 1 h at room temperature. Following washing with 0.2% BSA, the embryos were incubated for 3 min in the mounting medium with DAPI.
Immunostaining was performed with Nanog (Cell Signaling Technology, 1:500) and Cdx2 primary antibodies (Biogenex, 1:200). The secondary antibodies used in this research were as follows: Alexa594 goat anti-rabbit IgG, Alexa488 goat anti-rabbit IgG, and Alexa594 donkey anti-mouse IgG antibodies (Invitrogen, 1:500).

Rats and control mice were euthanized with an overdose of carbon dioxide and transcardially perfused with PBS. Brain tissue was subsequently harvested and post-fixed overnight in 4% PFA in PBS. Brains were cut in serial sections of 40 μm thickness with a vibrating microtome (Leica). For each sample, six series of sections were sequentially collected in free-floating conditions and permeabilized for 30 min with PBST (0.2% Triton X-100 in PBS) and blocked for 2 h with 5% normal donkey serum in PBST. Antigen retrieval was performed by boiling the sections for 1 min in 10 mM sodium citrate (pH 6) in a microwave. After three rinses with 0.1% Tween 20 in PBS, sections were incubated for 20 min with 10 μM X34 (Sigma) in 0.1% NaOH made in 40% Ethanol washed and incubated overnight at 4 °C with primary antibodies against Aβ: 82E1 (IBL, 1/150) or 6E10 (BioLegend, 1/200). The antibody stained sections were washed three times with 0.2% TritonX100-PBS and incubated with Alexa594 Donkey anti-mouse IgG (Thermo Fisher, 1/300) for 2 h at RT. After final washes with 0.2% TritonX100-PBS, sections were counterstained with TO-PRO (Thermo Fisher, 1/1000) and after three final washes were mounted on super frost microscope slides. Sections were visualized on a Nikon A1R Eclipse confocal system.