Maxiscript in vitro transcription kit

1 μg of plasmid containing full length UCP2 with 5′ and 3′ UTR’s (1–1647 nt) was linearized with Eco RV. The linearized plasmid was purified using acetate/ethanol precipitation. UCP2 was in vitro transcribed using MAXIscript in vitro transcription kit (Ambion). Transcription was confirmed by gel electrophoresis and concentration was determined using an absorbance at 260 nm. The in vitro transcribed RNA was biotinylated using the Pierce RNA 3′-biotinylation kit from Thermo Scientific according to the manufacturer's instructions.

Biotin pulldowns were performed as previously described^{63 (link)}. Briefly, biotinylated probes were generated by amplifying the TNF, Nos2, and Gapdh 3′ UTRs from the respective pmirGLO plasmid using forward primers that contained a T7 RNA polymerase promoter sequence (Table S2). From this amplified DNA, biotinylated mRNAs were synthesized using the MAXIscript in vitro transcription kit (Ambion) per the manufacturer’s instructions, using a ratio of 1:5 biotin-labeled CTP to unlabeled CTP. HEK293 cells were transfected with 5 μg of pcDNA3-TTP using lipofectamine. After 24 hours, cells were lysed with PLB supplemented with protease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Lysates were cleared by centrifugation at 16,000 × g. Reactions containing 500 nM of biotinylated transcripts were mixed with 40 μg of TTP-transfected HEK293 cell lysate for 30 min at room temperature, then incubated with 15 μL streptavidin-dynabeads (Invitrogen) for 30 min, with periodic disruption for brief vortexing. The beads were washed twice with PBS with 500 mM NaCl, proteins were eluted by boiling in 1X SDS-PAGE loading buffer, and samples were separated by SDS-PAGE and analyzed by western blot using anti-TTP antibody (Millipore, ABE283, 1:500).

In situ hybridization (ISH) was performed on 16 μm coronal cryosectioned tissues as previously described. Antisense riboprobes were generated from full length cDNAs of slit1 (MMM1013-98685876), alcam (MMM1013-202762192), spon1 (MMM1013-202701079) and per1 (MMM1013-202764685; from Open Biosystems; Huntsville, AL, USA). Riboprobes were synthesized using digoxigenin (DIG)-labeled UTP (Roche, Mannheim, Germany) and the MAXIscript in vitro Transcription Kit (Ambion, Austin, TX, USA). Probes were hydrolyzed to 500 nt. Coronal brain sections were prepared and hybridized at 65°C as previously described (Su et al., 2010 (link)), and bound riboprobes were detected by horseradish peroxidase (POD)-conjugated anti-DIG antibodies and fluorescent staining with Tyramide Signal Amplification (TSA) systems (PerkinElmer, Shelton, CT, USA). Images were obtained on a Zeiss Axio Imager A2 fluorescent microscope or a Zeiss Examiner Z1 LSM 700 confocal microscope. A minimum of three animals per genotype, age and time were compared in ISH experiments.

To describe the expression pattern of C1qbp in the brain, the fresh tissue was quickly frozen on dry ice. In situ hybridization histochemistry was processed as described previously^{72 (link)}. Briefly, serial coronal sections (12 µm) were cut using a cryostat from bregma level +3.5 mm to −15 mm, mounted on positively charged slides (SuperfrostUltraPlus™; Thermo Fisher Scientific, Pittsburgh, PA, USA), dried, and stored at −80 °C until use. Antisense [35 S]UTP-labelled riboprobes were generated using T7 RNA polymerase of the MAXIscript in vitro transcription kit (Ambion, Austin, TX) from PCR-amplified fragments of the cDNA subcloned into TOPO TA vectors.
Tissue was prepared using an mRNA-locator Kit (Ambion) according to manufacturer’s instructions. For hybridization, we used 80 µl hybridization buffer and 1 million DPM of labelled probe per slide. Washing procedures included a 30 min incubation in RNase A, followed by decreasing concentrations of sodium-citrate buffer (pH = 7.4) at room temperature, and then at 65 °C. After drying, slides were dipped in NTB nuclear track emulsion (Eastman Kodak, Rochester, NY), stored for 3 weeks at 4 °C for autoradiography. Then, the slides were developed and fixed with Kodak Dektol developer and Kodak fixer, respectively, counterstained with Giemsa, dehydrated, and coverslipped with Cytoseal 60 (Stephens Scientific).