D5905

Brains were sliced with a vibratome (Leica, VT1000E) on a coronal plane into 40 μm. After between two 4 × 10 min rinses in PBS, endogenous peroxidases were neutralized during 30 min in PBS containing 3% H₂O₂. To block the non-specific site, we used PBS solution with 1% bovine serum albumin (BSA), 3% normal goat serum (NGS), and 0.2% Triton ×100 during 2H. c-fos immunolabeling was performed with a purified polyclonal rabbit IgG anti-human c-fos [anti c-fos (Ab-5) (4-17) rabbit pAb, CALBIOCHEM] diluted 1:20.000 in 1% BSA, 3% NGS, and 0.2% Triton x100 during 38H. After 4 × 10 min rinses in PBS, sections were incubated for 2H with secondary biotinylated antibody (Biotin Goat anti-rabbit IgG (H + L), INTERCHIM) diluted 1:2.000000 in 1% BSA, 3% NGS, and 0.2% Triton ×100 during 2H. After 4 × 10 min rinses in PBS, the staining was revealed using H₂O₂ and diaminobenzidine (D-5905, SIGMA) for 3 min. After rinsing, sections were flattened on SuperFrost glass slides (Menzel-Gläser, Braunschweig, Germany), dehydrated with xylene, and mounted with Eukitt solution.

Immunohistochemistry (IHC) for Ki-67, p-Akt, and E-cadherin was performed using rabbit monoclonal antibodies. Tumor tissues were deparaffinized with xylene twice for 5 min and rehydrated and rinsed with tap water for 10 min. They were incubated at 4°C with primary antibodies overnight. Immunoreactivity was visualized with an avidin-biotin peroxidase reaction (PK-4001, Vectastain ABC kit). The peroxidase reaction was developed using a 3,3′-diaminobenzidine tetrahydrochloride (D-5905, Sigma). The sections were counterstained with hematoxylin before being mounted.

To determine which nuclei in the hypothalamus were responsive to test-pellet consumption, we used c-Fos as a marker of neuronal excitation, as previously performed in a laboratory [26 (link)]. Brains were flash-frozen in methylbutane on dry ice and then sectioned at 30 microns using a freezing–sliding microtome. Sections were washed in potassium-phosphate-buffered saline (KPBS), blocked with 2% donkey serum in KPBS with 0.4% Triton x-100 in KPBS, and incubated with the primary antibody for c-Fos (1:2500, #ab190289, Abcam, Cambridge, MA, USA) overnight for two nights at 4 °C. The sections were then incubated with the anti-rabbit biotinylated secondary antibody (1:500, #BA1100, Vector Laboratories, Burlingame, CA, USA) for 60 min at room temperature, followed by A/B solution (1:600, #PK-6100, Vector Laboratories) for 60 min at room temperature. The final step was an incubation of 0.2 mg/mL DAB substrate (D5905, Sigma, Burbank, CA, USA) and hydrogen peroxide for 15 min at room temperature. Sections were then mounted on gelatin-coated slides.

To evaluate the presence of vessels in the neoformed pulps were performed immunohistochemical experiments [30] (link). Briefly, formalin-fixed paraffin-embedded tissue sections (3 μm-thick) were applied on previously silane-coated slides. After the deparaffinization, the sections were rehydrated in decrescent ethanol-graded series. We used 3% H₂O₂ solution (45 minutes) to block the endogenous peroxidase. Finally, we used Protein Block (X0909, Dako, Carpinteria, CA, US) (10 minutes) to avoid the non-specific binding.
Polyclonal rabbit anti-VEGFR2 (1:700; Sigma-Aldrich) primary antibody was applied over the slices and maintained in a humid chamber (4 °C) overnight. Next, the slices were incubated with the detection system based in Chain Polymer-Conjugated Technology (EnVision^®, Dako) for 30 minutes at room temperature (RT). Then, the immunostaining was revealed using a chromogenic substrate mixture (3,3′-Diaminobenzidine; D5905, Sigma-Aldrich). Finally, Harry's hematoxylin was used to counterstain the sections. For positive control, we used normal dental pulp from rats while in the negative control we suppressed the primary antibody during the procedures.

Adult mice were subjected to the surgical procedure described above, in order to transplant hMSCs into the deep cerebellar nuclei of the cerebellum. Five different post-transplant timepoints were determined to confirm hMSC survival in the mouse brain: 48 h, 1 week, 4 weeks, 6 weeks, and 8 weeks post-transplantation of hMSCs. At each timepoint, mice were deeply anaesthetized and transcardially perfused with PBS, followed by 4% PFA in PBS. The brains were post-fixed overnight in fixative solution and embedded in paraffin (EG1140H, Leica, Germany); 4 μm thick paraffin sections were processed via immunohistochemistry for human nuclear antigen (HNA; MAB1281, 1:150, Merck Millipore, Lisbon, Portugal), and then developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB) substrate (D5905, Sigma, Germany). Slice microphotographs (total 3–4 sections) were acquired using DPController software (Olympus, Spain) and a camera (Olympus DP70, Spain) attached to a motorized microscope (4E14135, Olympus BX61, Spain) using a 4 × 0.16 numerical aperture objective.

All virus titrations were performed using 12-well standard cell culture plates seeded with cells to reach 100% confluency upon infection. Cells were inoculated with 10-fold serial dilutions of the recovered sample and were rocked at 37°C for one hour after which the inoculum was removed and replaced with an overlay of 1 ml of 1% methyl cellulose (Sigma Catalog # M0512) mixed 1:1 with 2x MEM (20% FBS, 8 mM glutamine, 20 mM NEAA, 2% penicillin/streptomycin, 2 mM sodium pyruvate). Plates were placed in a 37°C incubator with 5% CO2 for 4 days. Cells were stained using 70% ethanol containing 1% wt/vol crystal violet. Plates were incubated for 15 minutes at 22°C after which the fixative was decanted. The plates were rinsed with cold water and dried overnight at room temperature. The titer of DENV was determined through a focus-forming unit assay. Briefly, cells were fixed and permealbilized using 1 ml of a 1:1 acetone/methanol solution with a 60 minute incubation at 4°C. Virus foci were detected using a specific mouse monoclonal antibody from hybridoma 2H2 (Millipore catalog #MAB8705), followed by a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Millipore catalog #AP124P), and developed using a 50mg tablet of 3,3’-Diaminobenzadine tetrahydrochloride (Sigma catalog # D5905) dissolved in 20mL PBS with 8uL 30% hydrogen peroxide.