Streptavidin alexa fluor 555

HA was detected using biotinylated Hyaluronan Binding Protein (bHABP; AMS Biotechnology, Abingdon, UK). WFA histochemistry was performed using biotinylated Wisteria floribunda agglutinin (bWFA; Sigma-Aldrich, St. Louis, MO, USA), a lectin that binds to N-acetylgalactosamine residues of CSPG-glycosaminoglycan chains and glycoproteins, as a marker of PNNs (Giamanco et al. 2010 (link); Hartig et al. 1992 (link)). After blocking, sections were incubated in a solution of bHABP or bWFA, both dissolved in PBS containing 1% BSA, overnight at 4 °C. Reactions were visualized by incubating the samples with Streptavidin AlexaFluor 555 (Life Technologies, Carlsbad, CA, USA) for 1 h, diluted in 1:1000, in PBS.

BDA (Thermo Fisher Scientific, Waltham, MA, USA, D1817) and fluorescent retrograde tracer cholera toxin B subunit conjugated to fluorophore Alexa 488 (CTB488, Thermo Fisher Scientific, Waltham, MA, USA, C34775) were used to label neurons that projected from the target regions. BDA and CTB488 were unilaterally delivered into the PPC and ACC of male C57BL6J mice using a glass micropipette at the following coordinates (in mm): (i) −2.0 AP, +1.5 ML from bregma, −0.5 DV from the brain surface for the PPC; (ii) +1.0 AP, +0.5 ML from bregma, −0.5 DV from the brain surface for the ACC. Mice were injected with 200 nl of 10% BDA in the PPC or 200 nl of 1% CTB488 in the ACC, and mice were perfused 7 days or 5 days after injection. BDA was visualized by staining with streptavidin-Alexa Fluor 555 (1:1000, Life Technologies, Carlsbad, CA, USA).

Antibodies used in this study are as follows. Goat anti-FSTL1 (ab11805; Abcam), rabbit anti-FSTL1 (Proteintech), rabbit anti-DIAPH1 (5486; Cell Signaling Technology), rabbit anti-KSRP (A302-22A; Bethyl Laboratories), mouse anti-KRT14 (clone LLO01), mouse anti-EGF (clone 10825; R&D Systems), mouse anti-TGFβ1 (Novacastra), rabbit anti-GFP (Covance), rabbit anti-cMyc (Sigma-Aldrich), and rabbit anti-Wnt7a (ab100792; Abcam). Streptavidin Alexa Fluor 555, chicken anti-goat Alexa Fluor 488, and donkey anti-rabbit/mouse Alexa Fluor 488/555 were from Molecular Probes. Rabbit anti-Arp3 (4738), phospho (9101), and total ERK antibodies (9102) were from Cell Signaling Technology. Rabbit anti-Arpin (ABT251) was purchased from Merck Millipore. Recombinant human FSTL1 (His-tagged) expressed and purified from mammalian cells was purchased from Thermo Fisher Scientific.

After treatment with WABI, MM1R cells were fixed in 4% (v/v) formaldehyde. Cells were stained with rhodamine–phalloidin (Molecular Probes, Thermo Fisher Scientific Inc., Waltham, MA, USA) to visualize the cellular morphology, Hoechst 33,258 (Sigma-Aldrich, St. Louis, MO, USA) to visualize the nucleus and streptavidin-Alexa Fluor555 (Molecular Probes, Thermo Scientific, MA, USA) to visualize biotinylated WA. To determine WABI colocalization with specific proteins (BTK), WABI-treated MM1R cells were immunostained with primary (anti-rabbit BTK, Cell Signaling Technology, mAb#8547) and corresponding labeled secondary antibodies (anti-rabbit Alexa Fluor-488, Molecular Probes, Thermo Scientific, MA, USA). Cells were then placed on glass slides and mounted with coverslips using Fluoromount^TM (Sigma-Aldrich, St. Louis, MO, US)Confocal imaging was carried out by a laser-scanning microscope equipped with a Plan-Apochromat 63X/1.40 Oil DIC objective lens and excitation wavelengths 405, 488, 561 and 640 nm (Zeiss LSM 800, Carl Zeiss, Germany). At least 20 different microscopic fields were analyzed for each sample using ZEN imaging software (Carl Zeiss). ZEN lite^TM (Carl Zeiss, Germany) was used to perform image reconstruction and presentation.