Alexa fluor 488

Primary antibodies: 1:250 mouse monoclonal anti-chymase (Abcam, #ab2377) 1:200 mouse monoclonal anti- tryptase (Abcam, #ab2378), 1:200 goat polyclonal anti-chymase (Abcam, #ab111239), 1:100 rat monoclonal anti-c-Kit (biotin) (Abcam, #ab25022), 1:300 rabbit polyclonal anti-collagen I (Abcam, #ab34710), 1:200 rabbit polyclonal anti- COX2 (Abcam, #ab15191), 1:200 mouse monoclonal anti-βIII-Tubulin (Millipore, #MAB1637), 1:100 rabbit polyclonal anti-SCF (Fisher Scientific, #PA520746), 1:400 mouse monoclonal anti-GFAP (Sigma, #G3893) and 1:100 rabbit polyclonal anti-CD45-PerCP (BioLegend, #103130). Secondary antibodies: 1:500 goat anti-rabbit- AlexaFluor488 or AlexaFluor546 (Thermo Fisher Scientific, #A11035 or #A11034), 1:500 goat anti-mouse-AlexaFluor488, AlexaFluor546 or AlexaFluor633 (Thermo Fisher Scientific, #A11029, #A11030, or #A21052), 1:500 donkey anti-goat- AlexaFluor488 (Thermo Fisher Scientific, #A11055) and 1:500 Streptavidin- AlexaFluor633 (Thermo Fisher Scientific, #S21375). NeuroTrace 530/615 red fluorescent Nissl stain (Thermo Fisher Scientific, #B34650) was also used for neuronal visualization.

The antibodies used in this study were anti-C1q (Abcam, ab182451, 1:500), anti-DppII (R&D Systems, AF3436, 1:500), anti-Foxp2 (Abcam, ab16046, 1:500), anti-Gfap (STEM CELL Technologies, 60128, 1:500), anti-Iba1 (WAKO, 019-19741, 1:500), anti-Lamp1 (BD Biosciences, 553792, 1:500), anti-Pgrn (R&D Systems, AF2420, 1:1000), TDP-43 (Proteintech, 10782-2-AP, 1:500), anti-Tuj1 (Neuromics, CH23005, 1:500), anti-Vgat (Synaptic Systems, 131011, 1:300), donkey anti-goat IgG (H + L) Alexa Fluor® 488 (Thermo Fisher Scientific, A-11006), donkey anti-rabbit IgG (H + L) Alexa Fluor® 647 (Thermo Fisher Scientific, A-31573), goat anti-rat IgG (H + L) Alexa Fluor® 647 (Thermo Fisher Scientific, A-12247), donkey anti-mouse IgG (H + L) Alexa Fluor® 488 (Thermo Fisher Scientific, A-21202), goat anti-mouse IgY (H + L) Alexa Fluor® 488 (Thermo Fisher Scientific, A-21449) (for immunofluorescence); Ctsd (R&D Systems, AF1029, 1:500), Lamp1 (BD Biosciences, 553792, 1:500), LC3-I/II (Cell Signaling, 2775, 1:1000), Pgrn (R&D Systems, AF2557, 1:100), TDP-43 (Proteintech, 10782-2-AP, 1:1000), p-TDP-43 (Cosmo Bio USA, CAC-TIP-PTD-P03, 1:500), Actin (Novus Biologicals, NB600-532, 1:10,000), donkey anti-goat IgG-HRP (R&D Systems, HAF109), donkey anti-sheep IgG-HRP (R&D Systems, HAF016), donkey anti-rat IgG-HRP (R&D Systems, HAF005), goat anti-rabbit IgG-HRP (for western blot).

The following antibodies and dilutions were used: Sox9 (#ab185966, abcam, Cambridge, UK) 1:5000 for western blot (WB); 1:500 for immunofluorescence (IF); Sox2 (#MAB2016, R&D Systems, Wiesbaden, Germany) 1:1000 (WB) and 1:250 (IF); Olig2 (#AF2418, R&D Systems) 1:10,000 (WB) and 1:5000 (IF); GAPDH (#CB1001, Calbiochem, Darmstadt, Germany) 1:20,000; CHK1 (#2360, Cell Signaling Technologies (CST), Frankfurt am Main, Germany) 1:1000; phosphoCHK1 (CST #2348), 1:1000; CHK2 (CST #2662S) 1:1000; Survivin (R&D #AF886) 1:1000; TP53BP1 (NB #100-304, Novus Biologicals, Wiesbaden, Germany) 1:1000; γH2AFXSer139 (#05-636, clone JBW301, Merck Millipore, Darmstadt, Germany) 1:1000; F(ab′)2-Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-11070, Thermo Fisher) 1:500; F(ab′)2-Goat anti-mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (A-11020, Thermo Fisher) 1:500; F(ab′)2-donkey anti-goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-11055, Thermo Fisher) 1:500; donkey anti-goat IgG (sc2042, Santa Cruz, Dallas, TX, USA) 1:10,000.

Actin was fluorescently labeled on the surface lysine-328, using Alexa Fluor 488, Alexa Fluor 568, or Alexa Fluor 647 NHS ester (Thermo Fisher Scientific), or ATTO 643 NHS ester (Atto-tec) as described in detail in (57 (link)). The Arp2/3 complex was fluorescently labeled using Alexa Fluor 488 or Alexa Fluor 568 C₅-maleimide (Thermo Fisher Scientific). The protein solution was prepared for labeling by performing a buffer exchange to remove dithiothreitol (DTT) from the solution. This was accomplished by passing the protein solution through a MicroBiospin 6 column (Bio-Rad). The exchange buffer contained 20 mM Hepes at pH 7.2, 0.2 mM MgCl₂, and 0.2 mM ATP. The buffer exchange was performed by centrifuging the sample at 1000g for 4 min. Next, a 10-fold excess of Alexa Fluor 488 (or Alexa Fluor 568) C₅-maleimide dissolved in dimethyl sulfoxide (DMSO) was added to the Arp2/3 complex solution and incubated on ice for 1 hour. The reaction was stopped by adding 1 mM DTT to the solution. To remove any unreacted excess dye, a MicroBiospin 6 column (Bio-Rad) was used by centrifugation at 1000g for 4 min. We obtained on average 3.5 Alexa dyes per the Arp2/3 complex.

The following primary antibodies were used for immunohistochemistry: rabbit anti-active caspase-3 (1:500; Abcam, Cambridge, United Kingdom), rabbit anti-DCX (1:500; Cell Signaling Technology, Danvers, MA), rabbit anti-glial fibrillary acidic protein (GFAP) (1:500; Agilent, Santa Clara, CA), goat anti-Olig2 (1:50; R&D Systems, Minneapolis, MN), rabbit anti-Iba1 (1:500; Fuji-Wako, Osaka, Japan), rabbit anti-Ki67 (1:500; Abcam), goat anti-MBP (1:500; Santa Cruz Biotechnology, Dallas, TX), rat anti-MBP (1:1,000; Abcam), mouse anti-NeuN (1:100; Merck-Millipore, Burlington, MA), and rat anti-NG2 (1:500; Abcam). The conjugated secondary antibodies were donkey anti-rabbit, anti-rat, anti-mouse, and anti-goat antibodies (Alexa Fluor 488, 568, and 647; Thermo Fisher Scientific, Waltham, MA) at a dilution of 1:500. For the EdU reaction, azide-modified dyes (Alexa Fluor 488 and 594 azide; Thermo Fisher Scientific) were used.