Anti-mouse or anti-goat (Life Technologies, Cat# A11001; Invitrogen, Cat# A11055) and
Anti-mouse or anti-goat (Life Technologies, Cat# A11001; Invitrogen, Cat# A11055) and Alexa Fluor 594 goat anti-rabbit secondary antibodies (Invitrogen, Cat# A11008) were added for 2 h to detect Iba1 and TLR2 followed by mounting of sections with DAPI (Invitrogen, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27486068 36935). Fluorescent images were acquired at room temperature on a Zeiss Observer Z1 inverted microscope (Carl Zeiss, German); images were processed using AxioVs 40 4.8.0.0 software (Carl Zeiss MicroImaging). Photographs were acquired using an AxioCam MRm digital camera (Carl Zeiss, German).RNA extraction, GW610742 web reverse transcription, and quantitative polymerase chain reaction (qPCR)were set up as follows: 10 l SYBR Green Mastermix, 0.5 l forward primers, 0.5 l reverse primers, and 9 l DEPC-treated H2O. Ninety-six-well plates were placed into a 7500 fast real-time PCR system (Applied Biosystems, Grand Island, NY). Mouse primers for TNF, IL-6, and MCP-1 were purchased from (Invitrogen, Mm00443258, Mm00446190, and Mm00441242).TNF and MCP-1 analyses by ELISASupernatant fractions collected from BV-2 cells that were treated with cocaine in the presence or absence of the indicated inhibitors or siRNA were examined for secreted TNF and MCP-1 protein levels using the commercially available ELISA kits (R D Systems, MTA00B and MJE00). The data presented represent results obtained from three independent experiments.Flow cytometryCells were stained for flow cytometry according to a previously published protocol, with some modifications [23]. After detaching from plates, BV-2 cells were washed once and resuspended in 1 ml of staining buffer (PBS with 2 FBS). Cells were counted and incubated with anti-CD16/CD32 (1 g/106 cells) to block FcII/III receptors. TLR2 antibody [T2.5] (FITC) (Abcam, Cat# ab59711) was added to the cells, and the mixtures were incubated for 10 min on ice in the dark. Cell suspensions were then exposed to direct fluorescent light for 15 min at room temperature. Following two washes with staining buffer, cells were fixed with 0.5 PFA. The cells were analyzed on an LSR II flow cytometer (BD Biosciences, San Jose, CA, USA) using FACSDiva software.Chromatin immunoprecipitation (ChIP) assayTotal RNA was extracted using Trizol reagent (Invitrogen, 15596-018). Briefly, monolayer cells in six-well plates were washed with PBS and lysed directly adding 1 ml Trizol. Cell lysate was aspirated into new 1.5 ml microcentrifuge tubes followed by addition of 0.2 ml of chloroform. After extensive mixing, the samples were centrifuged at 12,000g for 15 min at 4 . The upper aqueous phase was transferred to a new tube followed by addition of 500 l of isopropyl alcohol. Samples were incubated for 10 min and centrifuged again to precipitate total RNA. Total RNA was dissolved in DEPC-treated H2O and quantified. Reverse transcription reactions were performed using a Verso cDNA kit (Invitrogen, AB1453/B). The Reaction system (20 l) included 4 l 5 ?cDNA synthesis buffer, 2 l dNTP mix, 1 l RNA primer, 1 l RT enhancer, 1 l Verso enzyme Mix (Invitrogen, AB-1453/B), 1 g total RNA template, and a variable volume of water. Reaction conditions were set at 42 for 30 min. The qPCRs were performed by using SYBR Green ROX qPCR Mastermix (Qiagen, 330510). Reaction systemsThe ChIP assay was performed according to the manufacturer’s instructions (Upstate, Billerica, MA, USA) with slight modifications. After treatment of the cells, 18.5 fresh formaldehyde was added directly into the medium at a final concent.
