Alexa fluor 405

Single-cell suspensions were prepared from 4-day cultures of breast reduction samples, and Epithelial Cell Adhesion Molecule positive (EpCAM⁺) cells were immunomagnetically isolated (> 90% purity, Additional file 1: Figure S4C) as described before [1 (link)] and cells were stained with anti-α6 integrin (CD49f), conjugated to alexafluor 647 (clone# G0H3, Biolegend.), anti-CD90, conjugated to phycoerythrin (PE) (clone# 5E10, Biolegend), anti-NOTCH3 (clone# 603532, R&D systems), and anti-FZD7 (clone# 151143, R&D systems) antibodies. A goat anti-rat fluorescein isothiocyanate (FITC) was used to detect FZD7 protein, a goat anti-mouse Alexa Fluor 405 (Invitrogen) was used to detect the NR3 protein, and propidium iodide (PI) exclusion was used to identify live cells. The bulk bipotent progenitors (BP, EpCAM⁺CD49f⁺CD90⁺), BP-subset “a” (EpCAM⁺CD49f⁺CD90⁺ NR3^highFZD7⁺), BP-subset “b” (EpCAM⁺CD49f⁺CD90⁺NR3^−/lowFZD7⁺), BP-subset “ab” (EpCAM⁺CD49f⁺CD90⁺NR3^mediumFZD7⁺), bulk luminal-restricted progenitors (LRPs, EpCAM⁺CD49f⁺CD90⁻), LRP-subset “a” (EpCAM⁺CD49f⁺CD90⁻NR3⁺FZD7⁺), and the LRP-subset “b” (EpCAM⁺CD49f^l+CD90⁻NR3⁻FZD7⁺) were isolated via Fluorescent Activated Cell Sorting (FACS).

Tissues were fixed with whole-body 4% paraformaldehyde (PFA) perfusion, and the brains were incubated in 4% PFA for an additional 12–24 hr after dissection. Coronal slices 80 µm thick were made with a vibratome. Free-floating sections were stained for parvalbumin with Pvalb (Swant, Marley, Switzerland; PV27) primary antibody with Alexa Fluor 405 (Invitrogen, #A-31556) or Alexa Fluor 594 (Cell Signaling Technology, Danvers, MA; #8889) secondary antibodies.

30 μm-thick coronal brain sections were permeabilized with 0.3% Triton-X and incubated with NH₄Cl 50 mM for 30 min at room temperature. Blocking was performed with a solution of 2% bovine serum albumin and 3% fetal bovine serum for 2 h at room temperature. Afterward, sections were incubated overnight at 4°C with primary antibodies [rabbit polyclonal anti-IBA1 (1:1,000, Wako) and goat polyclonal anti-IL1β (1:500, R&D Systems)]. After washing, secondary antibodies [1:500 anti-rabbit Alexa Fluor 555, anti-goat Alexa Fluor 405 (Invitrogen)] were incubated for 1 h at room temperature. The sections were mounted with the Mowiol mounting medium.
For IBA1/IL1β quantification, 1024 × 1024 pixel confocal fluorescent image stacks from these brain sections were obtained with a TCS SP5 LEICA confocal microscope, using an X20 objective. We obtained pictures of the CA1 hippocampus and motor cortex. ImageJ software (https://imagej.nih.gov/ij/) was used for image quantification. N = 5–7 animals per group were analyzed, and three brain slices were analyzed per animal.

The following antibodies were used: rabbit anti-Asl (1:500, recognizes the N-terminus of the protein; Dzhindzhev et al., 2010 (link); Fu and Glover, 2012 (link)), chicken anti-Dplp (1:500; Rodrigues-Martins et al., 2007 (link)), rat anti-Sas6 (1:500, against GST-Sas6-236-472 aa; Dzhindzhev et al., 2014 (link)), rabbit anti-Ana1 (1:500, against His-Ana1-1400-1729 aa; Fu et al., 2016 (link)), guinea pig anti-Cep135 (1:500, against His-Cep135-810-1059 aa), rabbit anti-GFP (1:500; Fu et al., 2009 (link)), guinea pig or rabbit anti-Asl and rabbit anti-Cep97 (1:500, against His-Asl-1-300 aa and GST-Cep97-670-806 aa, respectively; serum produced by the Animal Facility, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and purified as previously described; Fu et al., 2016 (link)), mouse anti-acetylated tubulin (1:500, T7451; Sigma-Aldrich), mouse anti–phospho-histone H3 Ser10 (1:250, 9706; Cell Signaling Technology), GFP-booster Atto488 (1:200, ChromoTek), GFP-booster Atto647N (1:200, ChromoTek), and HRP-conjugated anti–β-tubulin (1:5,000, BE3312; Shenzhen Bioeasy Biotechnology). Secondary antibodies were conjugated with Alexa Fluor 405, 488, 568, or 647 (1:500; Invitrogen), with Abberior STAR RED (1:150; Abberior), and with HRP (1:10,000; Jackson ImmunoResearch). The fluorescent nanoparticles were from Abberior (1× Nanoparticles Red Fluor, 40 nm; NP-3004).

Donor cells were permeabilized with 0.1% saponin and 5% FBS for 20 min. After fixation, co-cultures were washed with 100 mM glycine in PBS and permeabilized/blocked in a PBS buffer containing 0.02% Triton X-100, 0.1% BSA, and 0.05% Tween-20, along with 10% goat serum (Sigma-Aldrich) and 1% goat anti-mouse IgG (Dako). The following primary antibodies were used: mouse anti-KDEL (marker for endoplasmic reticulum and cis-Golgi network, 1:400; Enzo Life Sciences), mouse anti-SV2 (Synaptic vesicle glycoprotein 2a, marker for the synapse and synaptic vesicles, 1:500; deposited by K.M. Buckley to the Developmental Studies Hybridoma Bank, The University of Iowa), mouse anti-TSG101 (tumor susceptibility gene 101, marker for multi-vesicular bodies (MSBs) and other endosomal compartments, 1:400; Thermo Pierce), mouse anti-VAMP2/synaptobrevin (Vesicle-associated membrane protein 2, marker for the synapse and synaptic vesicles, 1:100; Synaptic Systems), and mouse anti-LAMP2 (Lysosomal associated membrane protein 2, marker for lysosomes/endosomes, 1:100; Southern Biotechnology). Incubation with the indicated primary antibody (overnight, 4°C) was followed by incubation with a secondary antibody conjugated with either Alexa Fluor 405 or Alexa Fluor 488 (Invitrogen) (75 min for donor cells only or 45 min for co-culture, RT). Finally, slides were mounted with ProLong containing DAPI.

The following primary antibodies were used for immunofluorescence: rabbit anti-Asl (Conduit and Raff, 2010 (link)), guinea-pig anti-Asl (Roque et al., 2012 (link)), rat anti-Asl (Franz et al., 2013 (link)), mouse anti-GTU88* (Sigma-Aldrich), mouse anti-α-tubulin (DM1 α; Sigma-Aldrich), rabbit anti-Cnn (Lucas and Raff, 2007 (link)), sheep anti-Cnn (Conduit et al., 2014a (link)), rabbit anti-Ana1CT (Stevens et al., 2010a (link)), rabbit anti-Ana1Mid (Conduit and Raff, 2010 (link)), guinea-pig anti-Ana1 (Conduit et al., 2014b (link)), rat anti-Ana1CT (this study), rabbit anti-Sas4 (Basto et al., 2006 (link)) and rabbit anti-Spd-2 (Dix and Raff, 2007 (link)) antibodies, all used at 1:500 (see details in Table S1). Secondary antibodies conjugated to Alexa Fluor 405, 488, 568, 594 (used for SIM) and 647 (Invitrogen) were used, all at 1:1000; GFP-booster–atto488 (ChromoTek) was used at 1:500. DNA was labelled with Hoechst 33342 (Invitrogen). Rabbit anti-Ana1 (Conduit et al., 2010 (link)) and mouse anti-actin (Sigma-Aldrich) antibodies, and appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare) were used for western blotting (all 1:3000).

Polyclonal rabbit anti GAP45 (1:10,000)^{63 (link)}, anti HA (1:1,000, Sigma H6908), anti HSP70 (1:1,000)^{64 (link)}, anti CPN60 (1:3,000)^{65 (link)}, anti-catalase (1:2,000)^{66 (link)}, anti IMC1 (1:2,000)^{67 (link)}. Monoclonal mouse anti actin (1:20)^{68 (link)}, anti GRA1 and anti GRA3 (1:20, gifts of Dr. J. F. Dubremetz), anti myc (1:10, 9E10), anti Ty (1:10, BB2), anti P21 (1:10)^{49 (link)}, anti SAG1 (1:10, gift of Dr. J. F. Dubremetz). FITC conjugated lectin (DBA, 1:500, Vector Laboratories FL-1031-2). Secondary antibodies for immunofluorescence: anti mouse Alexa fluor 405 (1:3000, Invitrogen A31553), 488 (1:3000, Invitrogen A11001), 594 (1:3000, Invitrogen A11005), anti-rabbit Alexa fluor 488 (1:3000, Invitrogen A11008), 594 (1:3000, Invitrogen A11012). Secondary antibodies for western blot: anti mouse HRP (1:3000, Sigma A5278), anti-rabbit HRP (1:3000, Sigma A8275).