Anti mouse igg conjugated to alexa fluor 647

Rats were deeply anesthetized with Isoflurane and transcardially perfused with ice-cold PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains were fixed in PFA overnight and then transferred to 30% sucrose in PBS to equilibrate for 3 days as described previously (Lee et al., 2015 (link)). Twenty micrometer coronal sections were made with a cryostat and washed with PBS for 10 min. The sections were washed in PBS and coverslipped with Vectashield mounting medium. The sections were also made after viral transfer for opsin verification, and were stained with anti-rabbit GFP (1:500, Abcam, Cambridge, MA, USA #AB290), NeuN (1:200, Vector Laboratories, Burlingame, CA, USA), DAPI (Vector Laboratories, Burlingame, CA, USA), and CaMKII-α (6G9) mouse mAb (1:200, Cell Signaling Technology, Danvers, MA, USA, #50049) antibodies. Secondary antibodies were anti-rabbit IgG conjugated to AlexaFluor 488, and anti-mouse IgG conjugated to AlexaFluor 647 (1:500, Life Technologies, Carlsbad, CA, USA). Images were acquired with a Zeiss LSM 700 Confocal Microscope (Carl Zeiss, Thornwood, NY, USA).

Samples (1 cm segments of basal inflorescence stems) were immersed in a fixative solution (4% paraformaldehyde and 0.05% glutaraldehyde in 25 mM phosphate buffer, pH 7.2). After three washes in 25 mM phosphate buffer, pH 7.2, the segments were dissected into approximately 3-mm-long pieces, dehydrated in a gradient ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 99.5%) series, and embedded in LR White resin according to the manufacturer’s instructions (TAAB, Reading, UK). The incubation at each step was performed in a laboratory rotator. The immunolabeling procedures were performed according to [51 (link)] with modifications. Transverse 0.5-μm-thick sections were labeled with UX1 (1:3) and AX1 (1:20) primary antibody diluted in 1% BSA in 0.1 M phosphate buffer, pH 7.2. The secondary antibody was anti-mouse IgG conjugated to Alexa Fluor 647 (Life Technologies) diluted in 1:100 in 1% BSA and 0.05% Tween-20 in phosphate buffer, pH 7.2. The sections were mounted in Milli-Q water, and the fluorescence signal was observed using the excitation filter 646 nm and emission filter 664 nm.

Rats were deeply anesthetized with isoflurane and transcardially perfused with ice-cold phosphate-buffered saline (PBS) and paraformaldehyde (PFA). Brains were fixed in PFA overnight and then transferred to 30% sucrose in PBS to equilibrate for 3 days^{51 (link)}. Then, 20 µm coronal sections were collected using Microm HM525 Cryostat (Thermo Fisher Scientific), washed in PBS, and coverslipped with Vectashield mounting medium. Images containing electrodes or cannula were stained with cresyl violet or hematoxylin and eosin stain, and viewed and recorded under a Nikon eclipse 80i microscope with a DS-U2 camera head. Sections were also made after viral transfer for opsin verification, and these sections were stained with anti-rabbit GFP (1:500, Abcam, Cambridge, MA, #AB290), CaMKII-α (6G9) mouse monoclonal antibody (1:100, Cell Signaling Technology, Danvers, MA, USA #50049), and 4′,6-diamidino-2-phenylindole (DAPI; 1:200, Vector Laboratories, Burlingame, CA) antibodies. Secondary antibodies were anti-rabbit IgG conjugated to AlexaFluor 488, and anti-mouse IgG conjugated to AlexaFluor 647 (1:500, Life Technologies, Carlsbad, CA). Images were acquired with a Zeiss LSM 700 Confocal Microscope (Carl Zeiss, Thornwood, NY). Animals with improper fiber or electrode or cannula placements, low viral expression, or viral expression outside the ACC were excluded from further analysis.