Anti magl

Chemicals were of the purest analytical grade. MEM, glutamine, PES, DMSO, collagenase, DNase1, trypsin, BPA (Bisphenol A), ABTS (2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), and hyaluronidase were purchased from Sigma (St. Louis, MO, USA). Rabbit anti-CB₁, anti-CB₂, anti-GPR55, anti-FAAH, anti-DAGL-α, anti-DAGL-β, anti-MAGL, anti-NAPE-PLD polyclonal antibodies, and KT109 were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Mouse anti-actin antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and rabbit anti-TRPV1 polyclonal antibody was from OriGene Technologies (Rockville, MD, USA). Rabbit anti-inhibin B polyclonal antibody was obtained from Elabscience Biotechnology (Houston, TX, USA), and rabbit anti-transferrin polyclonal antibody was from Proteintech Group (Chicago, IL, USA). Iodoresinferatoxin was obtained from Tocris Cookson (Bristol, UK), whereas SR144528 was a kind gift from Sanofi-Synthelabo (Montpellier, France).

Cortical or hippocampal tissue was weighed, homogenized and incubated for 30 min in RIPA buffer containing protease inhibitors. The homogenate was then centrifuged at 13,000 g for 10 min at 4 °C. Protein samples were separated on premade 4 – 12% SDS-PAGE gels and were transferred to PVDF membranes (Bio-Rad). After incubation with primary antibodies, including anti-Aβ₄₂ (1:1,000, Invitrogen, USA), anti-BACE1 (1:200, Bioss, China), anti-APP (1:200, Bioss, China), anti-COX-2 (1:200, Cayman, USA), and anti-MAGL (1:200, Cayman, USA), overnight at 4 °C, the membranes were blotted with IR Dye 800 CW-conjugated secondary antibody (1:5000, LiCor Biosciences, USA) at room temperature for 1 h. The densitometry of the bands was scanned and measured using a LI-COR Odyssey Infrared Fluorescent System. The density of each band was quantified using Image-pro Express software, version 6.0 (Media Cybernetics, USA) and was normalized to the corresponding β-actin (1:1000, Cell Signaling, USA) value to account for variations in loading.

The chemicals used in this study were obtained from the following companies: secondary goat anti-mouse IgG conjugated to HRP (sc-2005) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP), (cat. 111-035-003) from Thermo Fisher Scientific (Waltham, MA, USA). The mouse-to-mouse HRP ready-to-use kit was obtained from ScyTek Laboratories, Inc. (Logan, UT, USA). All of the other reagents and ABTS (2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt) were purchased from Sigma (St. Louis, MO, USA) and were of the purest analytical grade. Rabbit anti-CB₁ (cat. 101500), anti-CB₂ (cat. 101550), anti-GPR55 (cat. 10224), anti-FAAH (cat. 101600), anti-MAGL (cat. 100035), and anti-NAPE-PLD (cat. 10305) polyclonal antibodies were obtained from Cayman Chemicals (Ann Arbor, MI, USA). Rabbit anti-DAGL-α (cat. PA5-23765) and anti-DAGL-β (PA5-26331) polyclonal antibodies were obtained from Invitrogen (Thermo Fisher Scientific; Waltham, MA, USA). The rabbit anti-TRPV1 (cat. TA336871) polyclonal antibody was obtained from OriGene Technologies (Rockville, MD, USA).