Alexa fluor 488 hydrazide

Electrodes were pulled using a horizontal puller (Sutter Instruments) using filamented, thin-wall glass (Sutter Instruments). Intracellular pipette solution consisted of the following (in mM): K-gluconate (120), KCl (20), HEPES (10), diTrisPhCr (7), Na₂ATP (4), MgCl₂ (2), Tris-GTP (0.3), EGTA (0.2) and buffered to pH 7.3 with KOH. To visualize electrodes, the cyan-green fluorescent dye Alexa Fluor 488 hydrazide (Thermo Fisher Scientific) was added to the intracellular electrode solution (0.3% weight/volume).
Electrode resistances were between 4 and 7 MΩ, with access resistance values between 15 and 38 MΩ. Seal resistance values were always greater than 2 GΩ. Capacitance was fully compensated in voltage clamp during the on-cell configuration prior to breaking into the cell. For current-clamp recordings, full bridge balance compensation was used. Series resistance compensation between 45–65% was used during voltage clamp recordings. Voltage trace signals were amplified and low-pass filtered at 10–20 kHz before being digitized at 20–50 kHz. For current traces, signals were low pass filtered at 4 kHz. All electrophysiology was carried out using a Multiclamp 700B (Molecular Devices) and a Digidata 1550 (Molecular Devices). Liquid junction potentials were not corrected.

Biotinylation and fluorescence labeling of aptamers was performed by 3′-end ribose oxidation using sodium metaperiodate followed by reaction with Alexa Fluor 488–Hydrazide or EZ-link Biotin-LC-Hydrazide using the standard protocol of the provider (Thermo Fisher Scientific). Cy5 hydrazide was obtained from Lumiprobe.

Human colonic mucosa tissue was washed with phosphate-buffered saline (PBS), immersed in 30% (w/v) sucrose in PBS, and frozen in O.C.T. compound (45833; Sakura Finetek, Torrance, CA) at −80°C overnight. Frozen sections were cut at a thickness of 10 μm using a cryostat (CM3050S; Leica Biosystems, Wetzlar, Germany). The frozen sections were washed 3 times with PBS and then pretreated with 0.5% periodic acid (HIO₄) solution (86171; Muto Pure Chemicals, Tokyo, Japan) at room temperature for 30 minutes (Figure 2B). Sections were washed 3 times with PBS and then incubated with FAM hydrazide, 5-isomer (50 μmol/L, BP-23934; BroadPharm, San Diego, CA), Alexa Fluor488 hydrazide (50 μmol/L, A10436; Thermo Fisher Scientific, Waltham, MA), BDP FL hydrazide (50 μmol/L, 11470; Lumiprobe, Hunt Valley, MD), or fluorescein (50 μmol/L, F0095; Tokyo Chemical Industry, Tokyo, Japan) in PBS at room temperature overnight. After additional PBS washes, the tissue sections were mounted with Fluoro-KEEPER antifade reagent with DAPI (12745-74; Nacalai Tesque, Kyoto, Japan) and imaged by confocal fluorescence microscopy.

Into the 20 µL of crude PURExpress product, 5 µL of 50 mM Alexa Fluor 488 hydrazide (Thermo Fisher) was added. The reaction mixture was incubated at 37°C for 14 h. 10 µL of mixture was obtained at the time point of 1 and 14 h. The reaction mixture was purified and characterized by the same methods described in the previous and next section.

To guide electrodes during patch clamp recordings, mice that express the red fluorescent protein tdTomato were used to visualize layer 2/3 excitatory pyramidal cells in somatosensory cortex. C57BL/6J background, CaMKIIa-Cre mice (The Jackson Laboratory, stock #005359; Tsien et al., 1996 (link)) were crossed with the lox-stop-lox tdTomato reporter mice (The Jackson Laboratory, stock #007914; Zariwala et al., 2011 (link)). CaMKIIa promoter in transgenic mice has been established to drive specific expression of fluorescent protein in layer 2/3 pyramidal cells in S1, with expression in ∼32% of pyramidal cells in layer 2/3 (Wang et al., 2013 (link)).
The electrode pipette was visualized using the cyan-green fluorescent dye Alexa Fluor 488 hydrazide (Thermo Fisher Scientific), which was added to the intracellular electrode solution (0.3% weight/volume). Imaging was performed using a two-photon imaging system (Thorlabs) with a mode-locked Ti:Sapphire laser (Chameleon Ultra II; Coherent) set to wavelengths between 920 nm and 950 nm, which was used to excite both the Alexa Fluor 488 and tdTomato using a 20×, NA 1.0 (Olympus) objective lens. Laser scanning was performed using resonant scanners and fluorescence was detected using two photomultiplier tubes (Hamamatsu) equipped with red and green filters to separate emission from Alexa Fluor 488 and tdTomato.