Alexa fluor 555 conjugated streptavidin

Biocytin hydrochloride (0.5%; Sigma) was added to the pipette solution for whole-cell patch clamp, and infused into CA1 pyramidal cells, after measurements of basal transmission for general health check for 2–3 min. Then, the slices were fixed with 4% formaldehyde at 4 °C overnight and permeablized with 0.5% Triton X-100 in 1 × PBS. Alexa Fluor 555-conjugated streptavidin (2 mg ml⁻¹, Invitrogen) in 1 × PBS was used for staining (1:300) for 1–2 h. Fluorescent images were acquired using confocal laser scanning microscope (LSM780, Zeiss).

To detect the axonal plasticity induced by lithium treatment, we traced axons of the lateral spinothalamic tract. Animals were anesthetized at 42 dpi and laminectomy was performed at the L2 level to expose the dorsal segment of the spinal cord (Caglayan et al., 2019 (link)). Under visual inspection, 1 μl biotinylated dextran amine (BDA, 10,000 MW; D1956; Invitrogen, Waltham, MA, USA) diluted in 0.01 M PBS was bilaterally injected 0.3 mm lateral to the midline and 0.7 mm deep to the dorsal surface using a Hamilton syringe connected to a glass tip. The syringe tip remained inside the injection area for 1 min to prevent backflow. After 14 days, animals were sacrificed by transcardial perfusion with 4% PFA and coronal sections were collected. Then, sections were washed with PBS, blocked with 10% normal goat serum in PBS for 1 h at room temperature, incubated with Alexa Fluor 555 conjugated streptavidin (S21391; Invitrogen) for 90 min at room temperature and analyzed using confocal microscopy (LSM780, Carl Zeiss). The number of axons crossing the rostral and caudal spinal cord segments were blindly counted in the lateral spinothalamic tract of the ipsilesional and contralesional hemicord in ROI measuring 62,500 μm².

BM-DCs were grown on Bio-coat coverslips (BD biosciences), fixed in 4% paraformaldehyde, permeabilized with 0.1% saponin and blocked with 5% goat serum. Samples were incubated with anti-CD11b-V450 (BD Biosciences, dilution 1:50), anti-CD14-biotin (ebioscience; dilution 1:50) and rabbit anti-EEA1 (Cell Signaling Technology; dilution 1:100). Secondary antibodies Alexa Fluor 555-conjugated Streptavidin and Alexa Fluor 488-conjugated goat-anti-rabbit IgG were both from Molecular Probes and were added at a dilution of 1:100. Samples were then analysed with a Leica SP5 confocal microscope. Colocalization spectra of each channel in the region of interest were analysed using the Lecia LAS AF software.

Upon biocytin filling during whole-cell recording, slices were fixed in 4% paraformaldehyde in phosphate buffer saline solution (PBS) at 4 °C O/N. After permeabilization with 0.3% Triton X-100, slices were incubated in Alexa Fluor 647 or Alexa Fluor 555-conjugated streptavidin (Molecular Probes, Eugene, OR, USA). Slices were re-sectioned using a freezing microtome (Microm, Waltham, MA, USA) to a thickness of 100 μm and subsequently mounted using Mowiol mounting medium (Calbiochem, San Diego, CA, USA). Streptavidin stained cells were imaged using Zeiss LSM710 or LSM800 confocal microscopes (20 × objective). After imaging morphologies were reconstructed and scholl analysis (with 20 μm intersections from the soma) was performed using Neurolucida (MBF Bioscience, Williston VT, USA).

We measured the relative concentrations of a total of 170 proteins (see Additional file 1, Supplementary Table S2) with antibody arrays (RayBiotech Inc., Peachtree Corners, GA, USA) according to the manufacturer’s instructions. Briefly, a custom glass-based antibody array targeting the 170 proteins of interest was built, 100 μl of diluted serum sample was added to each well, incubated overnight at 4°C, and then extensively washed. We then incubated the wells with biotin-conjugated antibodies specific to the different proteins. Membranes were developed with Alexa Fluor^® 555-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA). The slides were scanned with 532 nm excitation and extracted using an InnoScan^® 300 Scanner (Innopsys, Carbonne, France).