Visualizing Layer 2/3 Pyramidal Cells

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.