Rabbit anti p2x7

Cells were seeded in 6-well plates, followed by different treatments for 72 h. After that, protein was extracted in lysis buffer (RIPA : PMSF : PhosSTOP = 100 : 1 : 1) for 15 min on ice. The supernatant was collected. An equal amount of proteins of various samples were separated by 10% sodium deodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were blocked using 5% nonfat dry milk at room temperature and then incubated with primary antibodies. After being washed three times with PBS, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Then, chemiluminescent signal was assessed by chemiluminescence development kit by an imaging system. The quantification of band intensity was performed by Image Pro-Plus software, and the expression levels of proteins were normalized to β-actin as the integrated optical density (IOD) ratio. The primary antibodies used are rabbit anti-P2X₇ (1 : 200, Alomone Labs), anti-total ERK1/2 and anti-phospho-ERK1/2 MAPK (1 : 1000, Cell Signaling Technology), and anti-TNF-α (1 : 800, Abcam).

P2rx7-EGFP embryonic brains (E14.5 or E18.5) were fixed for 3 h in 4% paraformaldehyde. For production of free-floating vibratome sections, brains were embedded in 4% agarose solution, and serial sections were cut 100 µm-thick in sagittal or horizontal section planes (our horizontal plane was parallel to the optic tract, i.e., horizontal to the hypothalamus, diencephalon and rostral midbrain regions). Primary antibodies—chicken anti-GFP (1:500; Aves Labs, Davis, CA) and rabbit anti-P2X7 (1:100; Alomone Labs, Israel) were diluted in 0.1 M PBS containing 0.5% Triton X-100 (wt/vol.) and 2% BSA (wt/vol.). Secondary antibodies used were anti-Chicken Alexa Fluor 488 (1:400; Thermo Fisher Scientific) and anti-rabbit Alexa Fluor 546 (1:500; Thermo Fisher Scientific), and the sections were counterstained with DAPI (Sigma). This material was interpreted morphologically according to standard atlases of the mouse brain and the prosomeric morphologic brain model (Puelles et al. 2013 (link); Puelles and Rubenstein 2003 (link), 2015 (link)). All images were acquired on a Leica TCS SPE confocal microscope using the 5x, 10x, 40x and 63x W/IR objectives.

E9.5 (n = 4), E13.5 (n = 4), and E18.5 (n = 4) embryonic brains from P2rx7-EGFP reporter mice were lysed and homogenized for 1 h at 4 °C in lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 1% Nonidet P40, Complete™ Protease Inhibitor Cocktail Tablets (Roche Diagnostics), 1 mM sodium orthovanadate (Sigma) and 1.5 µM okadaic acid (Merck Life Science, Madrid, Spain), pH 7.4. Protein extracts (20 µg) were electrophoresed on a 10% Tris–Glycine SDS-PAGE gel and transferred to nitrocellulose membranes (Amersham GE, Barcelona, Spain) saturated with 5% non-fat dried milk or 3% BSA for 1 h at RT. Blots were incubated overnight at 4 °C with the following antisera: rabbit anti-P2X7 (1:1000, 70 KDa; Alomone labs, Jerusalem, Israel), rabbit anti-GFP (1:1000, 27 KDa; Thermo Fisher Scientific), and rabbit anti-GAPDH (1:10,000, 37 KDa; Sigma). Then, the blots were washed in PBS-Tween and incubated for 1 h at RT with goat anti-rabbit IgGs coupled to horseradish peroxidase at 1:5000 dilution (Dako Cytomation, Glostrup, Denmark). Proteins were visualized by enhanced chemoluminescence detection (Perkin Elmer, Houston, TX). Images were captured with an ImageQuant LAS 500 device (Amersham GE) and analysed using ImageQuant software (Amersham GE).