Alexa fluor 488 conjugated secondary antibody

Immunohistochemistry was performed as described previously7 (link). Briefly, mice were perfused transcardially with 0.9% NaCl containing 25 U/mL heparin, then 4% paraformaldehyde in tris-buffered saline (pH 7.4). Brains were removed, post-fixed overnight at 4 °C in the same fixative, and cryoprotected in a series of solutions (10%, 20%, and 30% sucrose in phosphate-buffered saline [PBS]). Frozen brains were coronally cut (40 μm thick). Sections were blocked with PBS containing 5% normal goat serum and 0.2% Triton X-100 for 30 min and incubated with anti-IP₃K-A (Santa Cruz Biotechnology, Dallas, TX), anti-NeuN, anti-GFAP, anti-CamKIIα (Invitrogen, Carlsbad, CA), anti-GAD67 (Millipore, Billerica, MA), or anti-c-Fos (Santa Cruz Biotechnology) antibodies overnight at 4 °C. After PBS washes, sections were incubated with Cy3, Cy5, or Alexa Fluor^® 488-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at room temperature. After three washes, sections were mounted with aqueous mounting medium (Biomeda, Foster City, CA) and observed on an Axiovert 200 Fluorescent microscope (Zeiss, Oberkochen, Germany).

Discs were dissected in M3 medium (Sigma), incubated with anti-E-cad (1:200, DCAD2, DSHB) or anti-Notch (1:50, C458.2H, DSHB) antibodies in M3 medium at 4°C for 1 h. Then, the antibody was washed out with cold M3 medium, and discs were incubated in fresh M3 medium with or without 200 µM chloroquine (C-6628, Sigma). Five discs per time point (four discs in control without chloroquine) were fixed in 4% formaldehyde in PBS after labelling and stained with Alexa-Fluor-488-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Quantification of vesicle numbers was performed with the 3D ObjectCounter Plugin in Fiji.

The following antibodies were used for immunofluorescence: anti-Vimentin (Cell Signaling Technology, Danvers, MA, USA, 5741, 1:100), anti-FAP-1 (Abcam, Cambridge, UK, ab53066, 1:100), anti-α-SMA (Dako, Fisher City, CA, USA, M0851, 1:100,), anti-CDX2 (Epitomics, 2475-1, 1:100), anti-CK7 (Cell Signaling Technology, 4465, 1:100), anti-EpCAM (Miltenyi Biotec, 130-098-793, 1:50), anti-CD133 (Abcam, ab16518, 1:50), Alexa Fluor488-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA, USA, 1:50), PE-conjugated secondary antibodies (Dako, 1:50), and Cy3-conjugated secondary antibodies (Santa Cruz Biotechnology, 1:50). The following antibodies were used for immunoblotting: anti-CD81 (Santa Cruz Biotechnology, SC-7637, 1:500), anti-GAPDH (Abcam, ab9484, 1:1,000), anti-CD133 (Miltenyi Biotec, 130-092-395, 1:250), anti-Nanog (Cell Signaling Technology, 4903, 1:1000), anti-OCT4 (Cell Signaling Technology, 2890, 1:1000), anti-EphB2 (R&D Systems, AF467, 1:2000), anti-ALDH (Santa Cruz Biotechnology, sc-374076, 1:1000). anti-phospho-β-catenin (Cell Signaling Technology, Ser 552, 9566 s, 1:500) anti-β-catenin (Cell Signaling Technology, 8480 s, 1:1,000), anti-Wnt3a (Santa Cruz Biotechnology, sc136136, 1:200), and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology 1:10,000).

Indirect immunofluorescence was performed as previously described [26 (link),90 (link)]. The primary antibodies used were anti-CD82 (11-559-C100, Exbio Praha, AS, Vestec, Czech Republic) and anti-E-cadherin (sc-8426) followed by appropriate Alexa Fluor 488-conjugated secondary antibodies (Jackson ImmunoResearch labs, Cambridgeshire, UK). Cells were counterstained with DRAQ5 (Invitrogen-Thermo Fisher Scientific) for nuclei visualisation. The stained cells were washed with PBS and mounted on glass slides. Images were collected on a Leica TCS-SP scanning confocal microscope with 63× objective lens.

Mice were killed by CO₂ anesthetization followed by cervical dislocation. Brains and spinal cords were carefully dissected and post fixation for at least one week was carried out in 4% phosphate-buffered formalin at 4 °C before the tissue was embedded in paraffin. Immunohistochemistry was performed on 4 μm paraffin sections as described previously [38 (link)]. The following antibodies were used: anti-Aβ antibody 24311 (1:500, [41 (link)]), IBA1 (1:500; #234004, Synaptic Systems) and GPNMB (1:500, Santa Cruz). Biotinylated secondary anti-rabbit, anti-guinea pig and anti-goat antibodies (1:200) were purchased from Dako or Jackson Immunoresearch. The staining was visualized using the avidin-biotin complex method with a VECTASTAIN kit (Vector Laboratories) and diaminobenzidine (DAB) as a chromogen providing a reddish-brown color with hematoxylin as nuclear counterstain.
For double-immunofluorescence staining, polyclonal goat anti-GPNMB antibody (1:500, AF2330, R&D Systems) was combined with IC16 (1:1000, against the N-terminus of Aβ), GFAP (1:500, #173004, Synaptic Systems), NeuN (1:300, MAB377, Millipore) and IBA1 (1:500, #234004, Synaptic Systems), respectively. The staining was visualized using Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibodies (1:750, Jackson Immunoresearch) and analyzed using a Nikon Eclipse Ti-E fluorescent microscope.

Cells were fixed with 4% (w/v) paraformaldehyde in PBS and permeabilized by incubation for 1 min with 0.5% Triton X-100. Then, cells were incubated with blocking solution (3% BSA in PBS) and incubated overnight at 4 °C with primary antibodies against collagen I (Abcam, Cambridge, UK, ab34710) and fibronectin (Abcam, Cambridge, UK, Ab2413). Fluorescent immunostaining was revealed with Alexa Fluor 488-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, USA). 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Thermo Fisher Scientific, D1306) was used to stain cell nuclei. The confocal images were captured by a confocal LEICA SP5-AOBS microscope with a 63X/1.4 NA oil-immersion objective.