Tyramine

Four-micrometer-thick tissue sections were mounted on positively charged barrier frame slides, dewaxed in xylenes, and rehydrated through an ethanol dilution series (100% to 25%). Tissue sections were digested with 5 μg/mL of proteinase K for 20 minutes at 37°C to facilitate probe penetration and exposure of miRNA species. To minimize nonspecific binding based on charge interactions, tissues were subjected to a brief acetylation reaction [66 mmol/L HCl, 0.66% acetic anhydride (v/v) and 1.5% triethanolamine (v/v) in RNase-free water]. Then, tissue sections were prehybridized at the hybridization temperature (see Supplementary Table S1 for details) for 30 minutes in prehybridization solution which consisted of 50% deionized formamide, 5× sodium chloride/sodium citrate buffer, 1× Denhardt’s solution, 500 μg/mL of yeast tRNA, and 0.01% Tween. The prehybridization solution was replaced with 200 μL of hybridization solution containing 10 pmol of the hapten-labeled LNA probe and tissues were incubated for 90 minutes at the hybridization temperature and washed thrice for 10 minutes in sodium chloride/sodium citrate buffer at the established stringency of sodium chloride/sodium citrate (see Supplementary Table S1 for details). At this point, tissue slides were loaded onto the Biogenex i6000 staining machine (BioGenex Laboratories, Inc.), which was programmed to dispense 400 μL of the appropriate reagent per step. Slides were treated with 3% H₂O₂ to inactivate endogenous peroxidase and block with 5% bovine serum albumin in PBS (w/v). Followed by primary and secondary antibody incubation in PBT [1% bovine serum albumin (w/v), 0.1% Tween 20 (v/v) in PBS] and washes in PBST [0.01% Tween 20 (v/v) in PBS], tyramine-conjugated fluorochrome was applied to the slide and the tyramide signal amplification (TSA) reaction was allowed to proceed for 10 to 30 minutes. Sequential TSA rounds for the detection of other miRNAs, noncoding RNAs, or proteins followed the same protocol. Finally, slides were washed extensively with PBST and mounted with antifading ProLong Gold Solution (Invitrogen) with or without 4′,6-diamidino-2-phenylindole (for nuclear counterstaining). Please see Supplementary Table S2 for a list of antibodies used and Supplementary Table S3 for the preparation of tyramide-conjugated fluorescent substrates.

Cyclic voltammograms were acquired with an EI-400 potentiostat used in two electrode mode and TH-1 software (ESA Inc, Chemsfold, MA, USA) written in LabVIEW (National Instruments, Austin, TX, USA). The waveform was generated and the voltammetric signal was acquired with an ADC/DAC card PCI-6251 (National Instruments). A PCI-6711 DAC board (National Instruments) was used to synchronize waveform application, data acquisition and TTL pulses for the flow injection valve. The output waveform was filtered with a low pass 2 kHz filter to eliminate digitization steps.
For electrochemical measurements, three triangular waveforms were used. The first waveform (referred to as the 1.0 V waveform) was a ramp from −0.4 V to 1.0 V and back to −0.4 V at a scan rate of 300 Vs⁻¹ repeated at 10 Hz with a rest potential of −0.4 V between scans. A second waveform (referred to as the 1.3 V waveform) was a ramp from −0.4 V to 1.3 V and back to −0.4 V at scan rate of 400 Vs⁻¹ repeated at 60 Hz, also with a rest potential of −0.4 V between scans. The third waveform (referred to as the 1.4 V waveform) was a ramp from −0.6 to 1.4 V and back to −0.6 V at scan rate of 400 Vs⁻¹ repeated at 60 Hz with a rest potential of −0.6 V between scans. The latter two extended waveforms were applied at a frequency of 60 Hz to intensify any oxidative etching effects and to reduce the duration of the electrochemical experiments. In each experiment background current was allowed to stabilize for 15 minutes 24 (link).
Evaluation of the adsorption of catechols employed the same waveforms but with a repetition frequency of 1 Hz. The charge was obtained by integrating the oxidation peak of the cyclic voltammogram as previously described 17 (link). The area of the electrode was calculated from microelectrode dimensions measured with an optical microscope. To account for contributions from diffusion, cyclic voltammograms were simulated with DigiSim software (Bioanalytical Systems Inc, West Lafayette, IN, USA) using kinetic parameters and diffusion coefficients reported before 30 ,31 . The amount of adsorbed analyte was obtained by subtracting the computed diffusional component from the measured charge and converting it to the number of moles using Faraday’s law.
For tyramine fouling experiments, the carbon fiber microelectrode was cycled for 15 minutes in buffer with the 1.3 V waveform at 60 Hz. The response to 500 nM dopamine in a flow injection system was monitored for 10 consecutive injections 3 minutes apart using the 1.0 V waveform for detection (10 Hz application frequency). Then, the electrode was purposely fouled by applying the 1.0 V waveform at 10 Hz in 15 mM solution of tyramine in PBS buffer for 15 minutes. Afterwards, the response to 500 nM dopamine was again evaluated with the 1.0 V waveform for 10 consecutive injections 3 minutes apart (10 Hz application frequency). The recovery of electrode sensitivity was evaluated by cycling the electrode with the 1.3 V waveform in PBS buffer for 15 minutes at 60 Hz followed by testing with 500 nM dopamine in the flow-injection system with the 1.0 waveform for 10 consecutive injections 3 minutes apart (10 Hz application frequency). The responses for the 10 consecutive injections for each condition were averaged and normalized to the pre-tyramine injections.
All potentials are reported versus a Ag/AgCl reference electrode. Electrochemical measurements were performed in a grounded Faraday cage.