4 6 diamidino 2 phenylindole (dapi)

Cultured neurons were fixed with ice-cold fixative (4% paraformaldehyde in 100 mM phosphate buffer, pH = 7.4) for 20 min and then permeabilized with PBS containing 0.1% TritonX-100 for 15 min at room temperature. Cells were then incubated with blocking solution (5% normal donkey serum in PBS) for at least 30 min at room temperature and were then incubated with the primary antibodies for 24 h at 4°C. For visualization, appropriate secondary antibodies conjugated to Alexa 546 or 488 (goat, 1:2,000) (Invitrogen) were used. Nuclear staining was performed with DAPI (DAKO). Confocal images were captured on a Zeiss LSM5 confocal system (Zeiss) and were assembled using Adobe Photoshop and Illustrator Software. Images were analyzed using MetaMorph Software (Molecular Dynamics). A single threshold was set for each staining condition to capture puncta that were clearly distinguishable and to minimize counting merged structures. For quantification of GAD67- and vGluT1-positive areas, total positive puncta area was measured in randomly selected fields from each group.

Cells were fixed in 100% ice-cold methanol at −20 °C for 10 min, washed three times with 1X PBS and further permeabilized in 0.3% Triton X-100 for 10 min. Following blocking in 10% normal goat serum (MP Biomedicals), cells were incubated with the primary antibody in blocking buffer at 4 °C overnight. The following antibodies were used for immunofluorescence: H4/H2AS1ph (ab177309, Abcam; 1:2000), Lamin A/C (ab238303, Abcam; 1:1000), H3K4me3 (ab8580, Abcam; 1 μg/ml) and H3K36me3 (ab9050, Abcam; 1 μg/ml). Next, cells were washed three times with 1X PBS and following incubation with Alexa Fluor 568 goat anti-rabbit (A11011, Thermo Fisher Scientific; 1:1000) and Alexa Fluor 488 goat anti-mouse (A11001, Thermo Fisher Scientific; 1:1000) secondary antibodies diluted in 10% normal goat serum for 1 h at room temperature, nuclei were stained with DAPI (Dako) or Hoechst 33342 (Invitrogen). Samples were imaged on a ZeissAxio Observer.A1 microscope. For confocal and super resolution microscopy imaging was carried out on a ZEISS LSM 900 with Airyscan 2 using Zen blue for acquisition and processing. Airyscan2 images were processed using the default deconvolution settings and histogram stretching, applied when required, was identical between control and treated samples for each channel.

MSC-laden constructs were harvested on d1 and d21 and fixed in 3.7% PBS-buffered formaldehyde over night at 4 °C. TissueTek^® O.C.T. (Sakura Finetek, Torrance, LA, USA) was used for embedding and 8 µm cryo-sections were performed at a cryostat (CM 3050S, Leica, Wetzlar, Germany). GAG deposition was visualized with safranin O (counterstain: Weigert’s hematoxylin and fast green), and collagen deposition with picrosirius red (counterstain: Weigert’s hematoxylin) [60 (link),61 (link)]. Immunohistochemical stainings were performed as previously described [38 (link)]. Used antibodies were: anti-aggrecan, 1:300, 969D4D11, Thermo Scientific, Waltham, MA, USA; anti-collagen I, 1:200, ab34710, Abcam, Cambridge, UK; anti-collagen II, 1:1000, II-4C11, Abnova, Taipei, Taiwan; and goat-anti-mouse Alexa488, 1:400, 111-545-003, Jackson ImmunoResearch, Cambridge, UK); all in 1% BSA in PBS. Cells were counterstained with DAPI during mounting (Dako, Hamburg, Germany).

Sections of tumor microarray and breast cancer tissue resected from mice were deparaffinized and rehydrated in routine series. Antigen retrieval was performed with IHC-Tek epitope retrieval solution (IHC World, Woodstock, MD, USA) in a humidified-heated chamber. Sections were incubated overnight at 4 °C with antibodies to SPIN90 (lab-made), α-SMA (CST), Vimentin (clone V9, sc-6260, Santa Cruz, Dallas, TX, USA), and E-cadherin (clone G10, sc-88426, Santa Cruz) overnight at 4 °C. The nuclei were counterstained with DAPI or Mayer’s hematoxylin (Dako, Santa Clara, USA), and the stained area was observed using a confocal microscope (FV1000; Olympus, Tokyo, Japan), research slide scanner (Olympus), or Aperio ImageScope (Leica, Wetzlar, Germany).

Immunofluorescent staining was performed on the slides coated with monolayer cells. After being fixed in 4% PFA and permeabilized in 0.2% Triton X-100, the cells were blocked in 10% normal goat serum for 1 h and then incubated with primary antibody at 4 °C overnight. The slides of the mounting cells were subsequently incubated with Alexa Fluor 488 secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. After being counter stained with DAPI (Invitrogen, Carlsbad, CA, USA) for 5 min, the slides were observed under a fluorescent microscope. For MNNG/HOS-OSCs, the cells were collected and centrifuged at 1500 rpm for 5 min, and cell pellets were fixed in 4% PFA followed by immunofluorescent staining procedures. After being counter stained with DAPI, the cells were suspended and mounted using a fluorescence mounting medium (Dako, Glostrup, Denmark) on the slide. Images were acquired using a fluorescence microscope.

MSCs were fixed with 4% paraformaldehyde solution (Panreac) at room temperature for 10 min and incubated with 0.2% Triton ×100 (Sigma, St. Louis, MO, USA) solution at RT for 10 min (except neurotrimin labelling). Furthermore, MSCs were incubated for 1 h in 1% bovine serum albumin (BSA, Sigma) and 10% normal goat serum (Abcam, Cambridge, UK) solution at room temperature to block the non-specific interaction of antibodies. Subsequently, the samples were incubated with primary polyclonal rabbit antibody for αSMA (Biolegend, San Diego, CA, USA, 904601), perilipin (Thermo Fisher Scientific, PA1-1051), CHD3 (Cloud-Clone Corp., Wuhan, China, PAA317Mu01), neurotrimin (Affinity Biosciences, Melbourne, Victoria, Australia, DF4245), RDH10 (Affinity Biosciences, DF12105), or rabbit polyclonal IgG (Biolegend, 910801) in 1% BSA solution at +4° overnight. Then, the samples were incubated with fluorescence-labeled goat anti-rabbit or goat anti-mouse (Invitrogen, A-11001) secondary antibodies (A11034, Invitrogen) at room temperature for 1 h. Cell nuclei were labeled with DAPI (DAKO, Glostrup, Denmark). Samples were analyzed with a Leica DM6000B fluorescent microscope equipped with a Leica DFC 360FX camera (Leica Microsystems GmbH, Wetzlar, Germany) using the LasX program. The percentage of CHD3+ MSCs was evaluated in FIJI using IgG-based thresholding.