Goat anti rabbit alexa 488 secondary antibody

After recordings, rats were deeply anesthetized and perfused transcardially with cold 0.1 M sodium phosphate buffer (PB) followed by 4% paraformaldehyde. The left barrel cortex was cut tangentially in 100 μm sections to the white matter. The rest of the left hemisphere was sliced coronally in 100 μm sections. Tangential sections were stained with streptavidin conjugated to Alexa 594 (Life Technologies) to visualize recorded neurons. In tangential and coronal sections, ChR2-eYFP signal was amplified by using a rabbit anti-GFP primary antibody (at 1:1000, incubated overnight in 5% normal goat serum and 1% Triton-X in PB at 4˚C) and a goat anti-rabbit-Alexa488 secondary antibody (at 1:200, for 2 hr in 5% normal goat serum and 1% Triton-X in PB at room temperature, Invitrogen). Using epifluorescence or confocal microscopy, we confirmed the presence of infected somata in the targeted brain region and infected axons near recorded S1 neurons.
The location of a L2/3 cell relative to its barrel center was measured by 3D reconstruction in Neurolucida (MicroBrightfield). The radial trunk axons of the recorded L2/3 neurons were visibly well filled in the L4 sections. Its location was marked, and the borders of the L4 barrel it passed through were traced. The horizontal distance between the axon and the centroid of the barrel borders were then measured in the same section.

Cells (2.5×10⁴) were plated on 0.1% gelatin-coated coverslips in a 24-well plate and were maintained in culture for 24 h. Then, coverslips were washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and blocked with 1% goat serum for 1 h at room temperature. Primary antibodies used were rabbit anti-tubulin (1:100 concentration; Abcam, Cambridge, UK) and phalloidin-TRITC (1:100 dilution; Sigma-Aldrich). Antibody staining was carried out in antibody dilution buffer overnight at 4°C. For tubulin staining, after washing with PBS, a goat anti-rabbit Alexa488 secondary antibody was added (1:400 dilution; Invitrogen, Life Technologies, Carlsbad, CA, USA) for 1 h in the dark at room temperature. Coverslips were co-stained with DAPI (300 nM; Sigma-Aldrich) for 10 min at room temperature and mounted with ProLong Antifade reagent (Invitrogen) on glass slides. Images were observed with a Leica DM2000 LED (Leica Microsystems, Wetzlar, Germany).

Stage 42 to stage 47 tadpoles were euthanized with tricaine methanesulfonate and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.5, for 2 h. For coronal sections, tadpoles were cryoprotected in 30% sucrose overnight and embedded in OCT compound (Sakura Finetek, Torrance, CA, USA), and 25-μm cryostat sections were obtained. Coronal sections at the level of the optic tectum were incubated with a rabbit polyclonal antibody against a synthetic peptide from the N-terminal extracellular region of human CB₁ receptor (1:200 dilution; Cayman Chemicals) or a rabbit polyclonal antibody against the cannabinoid receptor CB₁(1–77) (1:250 dilution, Cat# 209550, Calbiochem). CB₁R primary antibodies were visualized using goat anti-rabbit Alexa 488 secondary antibodies (1:500 dilution; Invitrogen, Eugene, OR, USA). The specificity of CB₁R antibodies (1:500 dilution) to recognize endogenous Xenopus CB₁R was previously tested and confirmed by Western blot analysis: a band of∼60 kDa was detected by anti- CB₁R antibodies in stage 45 Xenopus brain lysates similar to the chick brain (da Silva Sampaio et al., 2018 (link)).