[IF=3.57] Wen et al. Striatal and Tegmental Neurons Code Critical Signals for Temporal-Difference Learning of State Value in Domestic Chicks. (2016) Front.Neurosci. 10:476  IF(IHC-F) ;  Chicken.  

PubMed:27877100

Sample:
Lane 1: Mouse Cerebrum tissue lysates
Lane 2: Mouse Cerebellum tissue lysates
Lane 3: Mouse Hippocampus tissue lysates
Lane 4: Rat Cerebrum tissue lysates
Lane 5: Rat Cerebellum tissue lysates
Lane 6: Rat Hippocampus tissue lysates
Lane 7: Human SH-SY5Y cell lysates
Lane 8: Human U87MG cell lysates
Lane 9: Human HeLa cell lysates
Lane 10: Human Jurkat cell lysates
Primary: Anti-GAD65 (bs-0400R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 65 kDa
Observed band size: 62 kDa

SHSY5Y cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (GAD65) polyclonal Antibody, Unconjugated (bs-0400R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Molt-4 cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 0.1% PBST for 20 min at room temperature, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with GAD65 Antibody(bs-0400R)at 1:100 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).

Blank control (Black line): Molt4 (Black).
Primary Antibody (green line):Rabbit Anti-GAD65 antibody (bs-0400R)
Dilution: 3μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647
Dilution: 3μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with PBST for 20 min at room temperature. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.