Formalin 10

PDOs were harvested out of matrigel by inoculating them with 1 mL Cell Recovery Solution (Corning Inc) for 60 minutes at 4°C. Organoids were then collected in cold phosphate-buffered saline, pelleted, and fixed in formalin 10% (Sigma-Aldrich) for 60 minutes. Following fixation, organoids were washed and resuspended in 200 μL of warm agarose 2%. The agarose pellet was dehydrated using ethanol and embedded in paraffin using a standard histologic protocol.

The generated matrices were fixed right after decellularization with formalin 10% (Sigma-Aldrich) for 20 min. Then, samples were treated with a solution of ammonium chloride 50 mM in PBS (NH₄Cl) (Sigma-Aldrich) to reduce the background due to aldehyde groups. Blocking and permeabilization was performed with a solution of 2% BSA and 0.2% Triton X-100 in PBS for 10 min at room temperature. Next, the matrices were stained overnight at 4°C with a combination of rabbit polyclonal anti-Fibronectin antibody (ab2413, Abcam) (1:200) and mouse monoclonal anti-Fibrillin-1 antibody (MAB2499 Millipore) (1:200), either mouse monoclonal anti-Collagen VI antibody (MAB3303, Millipore) (1:400) in PBS with 2% albumin from bovine serum (BSA, Sigma-Aldrich). On the next day, Alexa 568 goat anti-rabbit (A11036, Thermo Fisher Scientific) (1:1000) and Alexa 488 goat anti-mouse (A10667, Thermo Fisher Scientific) (1:1000) were used as a secondary antibody. The incubations were performed for 1 h at room temperature in PBS with 2% BSA. Samples were washed with PBS and mounted with Fluoromount (Sigma-Aldrich) on glass slides. Phalloidin−tetramethyl rhodamine B isothiocyanate (Sigma-Aldrich) was used for F-actin staining (1:1000). Nuclei were stained with Hoechst 33,342 (Molecular Probes) (1:1000). Both dyes were incubated for 1 h at room temperature.

Sodium orthovanadate (Na₃VO₄), trypsin inhibitor, PMSF, Nonidet P-40, Triton X-100, formalin 10%, aprotinin and leupeptin were obtained from Sigma-Aldrich Canada (Oakville, ON, Canada) and the Western Lightning Chemiluminescence Plus from PerkinElmer (Guelph, ON, Canada). Fetal bovine serum (FBS) and Dulbeco’s modified Eagle’s medium (DMEM) were purchased from Wisent Bioproducts (St-Bruno, QC, Canada). And Tween 20 as well as hydrogen peroxide (30%) from Fischer Scientific (Ottawa, ON, Canada). Polyethylenimine (PEI) was obtained from VWR (Mississauga, ON, Canada) and slowFade^TM Gold antifade reagent from Thermo Fisher Scientific (Waltham, MA, USA).

For γH2AX immunofluorescence staining, 5x10⁴ cells were seeded on glass slides (Thermo Fisher Scientific) and incubated overnight before adding treatments (Doxorubicin, carboplatin, olaparib). Forty-eight hr later, cells were fixed with formalin 10% (Sigma) for 10 min at room temperature, washed with PBS 1 X and then permeabilized with 0.25% Triton-X100 (Sigma) in PBS 1 X for 15 min. Slides were incubated 1 hr with DAKO blocking solution (Agilent) and incubated overnight at 4 C with anti-phospo-H2AX (1:2000). Cells were then washed three times with PBS 1X-Tween 0.05% followed by incubation with the secondary antibody (1:800; donkey anti-mouse IgG Alexa Fluor 488; Thermo Fisher Scientific). The glass slides were stained with DAPI, washed, and mounted with Fluoromount aqueous mounting medium (Sigma). Stained sections were scanned (Leica) by the molecular pathology core of the CRCHUM and photographs were analyzed using Visomorph viewer software for automatic counting of γH2AX foci.

Two 4t1 tumor mouse models (prepared as mentioned previously) were injected intravenously in their tail vein with Apt-ALGDG2-Iohexol (concentration: 1.6 µM as described above) every 18 hours for 48 hours. Two 4t1 tumor mouse models were used as controls without injection. Subsequently, each mouse was sacrificed and the chest cavity was opened with dissecting equipment to expose the internal organs. Afterward, the animal’s kidney, spleen, liver and tumor tissues were harvested for histopathological examinations. Tissues were fixed in formalin 10% (Merck, Germany) for 72 h and then dehydrated through a graded-alcohol series (70, 80, 90, 95 and 100%). Then, tissues were cleaned in two changes of xylene and impregnated with two changes of molten paraffin wax. The samples were embedded and blocked out in paraffin and sectioned at 5 μm slides. After sectioning, the slides were stained with hematoxylin and eosin (H&E). slides were monitored under light microscope and the photomicrographs of them were obtained for evaluation of tissue degeneration to assess the toxic effects of the novel nano-theranostics on the animal’s body and targeted nature of it to the tumor site.

A fraction of lung tissue was collected and fixed in formalin 10% (Merck, Darmstadt, Germany). The fragments were embedded in paraffin, and 4 to 5 µm sections were cut and stained with hematoxylin-eosin (H.E.). The images were acquired with an inverted microscope (Zeiss, Primovert, Gottingen, Germany) coupled to a digital camera system (Axiocam 105 color, Zeiss, Oberkochen, Germany) and processed by the Zeiss Software (Zen core, Oberkochen, Germany) in the Laboratory of the immunobiology of interaction Leishmania-macrophages of Instituto de Ciências Biomédicas da Universidade de São Paulo.