Alexa fluor 488 and 546 conjugated secondary antibodies

Rats were deeply anesthetized with chloral hydrate (400 mg kg⁻¹, i.p.) and perfused transcardially with 300 ml ice-cold PBS, followed by 200 ml 4% paraformaldehyde. The brain was then quickly removed from the skull and post-fixed in the same fixative for 24 h. The fixed brain was transferred to 30% sucrose solution in PBS with pH 7.4 until it sank, then embedded in a tissue-freezing medium (OCT) and stored at −30 °C. Coronal sections (20 μm) of the M1 were cut by a freezing microtome (CS1031L9705, Shandon, UK).
For immunohistochemistry, the primary antibodies used included anti-Cux1 (CDP, rabbit, 1:100, Santa Cruz Biotechnology, catalogue No: M-222), anti-VGlut2 (Guinea Pig, 1:5,000, Millipore, catalogue No: AB2251), anti-TH (rabbit, 1:500, Millipore, catalogue No: AB152), and anti-NeuN (mouse, 1:500, Millipore, catalogue No: MAB377). Sections were incubated in primary antibodies at 4 °C overnight, and stained with the secondary antibodies. To identify the laminar structure of the M1, the layer specific marker Cux1 (labels L2/3) (ref. 75 (link)) and VGlut2 (labels L1, L2/3b, L5b) were doubly stained using Alexa Fluor 488 and 546 conjugated secondary antibodies (Invitrogen Corporation). The exact stimulating and recording site were confirmed by verifying the depth of electrode track with reference to the thickness of each cortical layer.

MDA-MB-231 cells were plated on poly-d-lysine-coated coverslips in 12-well culture plates and incubated for 24 h at 37°C. Then, cells were fixed with methanol for 10 min at -20°C, permeabilized with 0.2% Triton X-100 in PBS for 10 min and blocked with 3% BSA in PBS. Following PKCζ antibody (Abcam, UK, ab59364; 1:50) and G3BP1 antibody (Santa Cruz Biotechnology, sc-365338; 1:10) incubation overnight at 4°C, the cells were stained with Alexa-Fluor 488 and 546 conjugated secondary antibodies (Invitrogen, United States) for 1 h at room temperature. Fluorescent signals were captured by a confocal microscope.