Alexa fluor 594 anti goat igg

Immunofluorescence staining was performed as described previously [29 (link)]. Briefly, the brain tissue was sampled as described above. The 15-25 μm brain sections were obtained, incubated with phosphate buffered saline (PBS) solution comprising 0.1% Triton X-100 for 30 min, and blocked with PBS solution comprising 5% goat serum (16210064, Gibco, NY, USA) for 30 min. Then, the sections were incubated overnight at 4°C with primary antibodies: IBA1 (ab5076, 1 : 500, Abcam, Cambrige, USA) and CD68 (ab6640, 1 : 200, Abcam, Cambrige, USA). After incubation, the sections were rinsed in PBS for 3 × 5 min washes and incubated for 1 h at 25°C with corresponding secondary antibodies: Alexa Fluor 488 antirabbit IgG (A11034, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 594 antigoat IgG (A11058, Invitrogen, Carlsbad, CA, USA). Lastly, the sections were dyed in DAPI solution (S36939; Invitrogen, Carlsbad, CA, USA) for 15 min at 25°C. The images of all sections were captured blindly using an A1 Si confocal microscope (Nikon, Japan).

After 2–28 days of growth primary neurons were fixed in 4% paraformaldehyde (PFA) in PBS, washed three times x 5 minutes in PBS, incubated over night at 4°C in PBS containing 10% sucrose and then frozen at -20 until used. The cells were then washed in PBS and blocked in blocking buffer (PBS pH 7.2 containing 0.25% BSA and 0.25% Triton-X-100) for 1hr at room temperature. The primary antibodies, anti-ZNF804A (D14, Santa Cruz) diluted 1:50 and doublecortin (Abcam) diluted 1:2000 in blocking buffer, were incubated over night at 4°C. The specificity of the anti-ZNF804A antibody was verified by Western blot of ZNF804A transfected HEK cells, as others have done previously [23 (link)]. Following primary antibody incubation, cultures were washed in PBS containing 0.25% Triton-X-100 and incubated with secondary antibodies (Alexa Fluor 594 anti-goat IgG and Alexa Fluor 488 anti-rabbit both diluted 1:600, Invitrogen) diluted in blocking buffer. Controls were made with only secondary antibody to avoid investigating unspecific labelling of cells. The slides were then washed twice and mounted in DTG mounting media (2.5% DABCO (Sigma-Aldrich), 50 mM Tris-HCl pH 8.0, 90% glycerol) with or without 0.375 mg/ml DAPI (Sigma-Aldrich). Culture slides were analysed at Olympus FV1000 confocal laser scanning microscope. Some cells were analyzed in three dimensions by confocal laser-scanning microscopy.

GPR56-expressing Colo320 cells were treated with 1 nmol/ml recombinant human progastrin for 30 minutes. Afterward, the cells were washed three times with PBS and fixed with 4% paraformaldehyde. After incubation with 2% BSA for 1 hour to block unspecific binding, the cells were incubated 30 minutes in prepared anti-progastrin antibody (Santa Cruz Biotechnology) and anti-GPR56 antibody (Santa Cruz Biotechnology). Cells were then washed and incubated with Alexa Fluor 594 anti-goat IgG (Invitrogen) and Alexa Fluor 488 anti-rabbit IgG (Invitrogen), and analyzed under a Nikon TE2000 microscope.

After a 72-hour post-SAH, mouse was sacrificed by an overdose of 2% pentobarbital sodium and underwent trans-cardiac perfusion with a 4% paraformaldehyde solution. Brain tissue was then removed and stored overnight in 4% paraformaldehyde. Dehydration was carried out using 30% sucrose solutions. Subsequently, brain sections measuring 15 μm were obtained and incubated in PBS solution containing 0.1% Triton X-100 for 30 min. To prevent non-specific binding, the sections were blocked with PBS solution containing 5% goat serum for an additional 30 min. Following this, the sections were incubated overnight at 4 °C with primary antibodies: IBA1 (ab289874, 1:300, Abcam) and CD68 (ab283654, 1:300, Abcam). Post-incubation, brain sections were thoroughly rinsed in PBS with three consecutive 5-minute washes. Subsequently, the sections were incubated at 25 °C for 1 h with corresponding secondary antibodies: Alexa Fluor 488 Anti-rabbit IgG (A11034, 1: 1000, Invitrogen) and Alexa Fluor 594 Anti-goat IgG (A11058, 1:1000, Invitrogen). Finally, the sections were stained with DAPI solution and observed using an A1 Si confocal microscope.