Faramount

Tissue sections were prepared as described above. Following de-paraffinization, sections were washed for 10 min in distilled water. All subsequent procedures with tissue preparations were carried out at room temperature [21 (link),22 (link)]. Endogenous peroxidase activity was blocked with hydrogen peroxide (1:10 w/PBS, 10 min). Thereafter tissue sections were treated with proteinase K (catalog no. S3020; DAKO, Carpentaria, CA, USA) for 10 min. After two washes in PBS, tissue preparations were treated with TritonX for 15 minutes, rinsed in PBS and incubated with anti-Caspase 3 antibody (1:100 in PBS; cat no. ab17815 abcam). After two washes in PBS, preparations were treated with biotinylated goat anti-rabbit Ig (catalog no. HK336-9R, BioGenex). Following a 5-min wash in PBS, slides were incubated for 20 min in peroxidase-conjugated streptavidin (catalog no. HK330-9k, BioGenex). Caspase 3 cells were visualized using 3-amino-9-ethylcarbazole chromogen (catalog no. HK121-5K Liquid AEC, BioGenex) for 3–5 minutes for optimal staining. Preparations were counterstained with hematoxylin (catalog no. 7221; Richard-Allan Scientific, Kalamazoo, MI, USA) and mounted in Faramount (catalog no. S3025, DAKO).

For immunocytochemistry, cultured adult neural stem cells were fixed with 4% paraformaldehyde for 10 min at RT (room temperature). The samples were washed twice with PBS (phosphate buffer saline, pH 7.4, Gibco). To permeabilize cells and block nonspecific binding, the samples were treated with 0.1% Triton X-100 (Sigma-Aldrich) and 3% bovine serum albumin (BSA, Sigma) in PBS for 2 hours at RT. Then, the samples were reacted at 4 °C overnight with diluted primary antibodies in PBS with 0.1% Triton X-100 and 3% BSA. The following dilutions of primary antibodies were used: for neuron staining, anti-Tuj-1 (1:500; Sigma), and for astrocyte staining, anti-glial fibrillary acidic protein (GFAP, 1:1000; Sigma). After rinsing 3 times in PBS, the samples were treated for 1 hour at RT with appropriate secondary antibodies: Alexa Fluor^® 594-conjugated (1:1000, Invitrogen) or Alexa Fluor^® 488-conjugated (1:500, Invitrogen). Nuclei were labeled with Hoechst 33342 (1:1000, Sigma). The stained samples were mounted on a slide glass with Faramount (Dako, Denmark).

Hydration was followed by antigen retrieval (Target Retrieval Solution, DAKO) and block of endogenous peroxidases. Application of goat serum (45’, 1/5, DAKO) blocks nonspecific sites. The first primary antibody was applied overnight (1/500, rabbit anti-α-SMA, Abcam ab5694) followed by incubation with Goat anti Rabbit IgG, conjugated with Horseradish peroxidase (45’, 1/100, DAKO P0448) containing 10% mouse serum (Sigma) and visualization by 3,3’-Diaminobenzidine (DAKO K346711). After thorough rinsing and block with donkey serum (45’, 1/5, Sigma), the second primary antibody is applied overnight (1/200, Goat anti-osteopontin, R&D AF808). The next day, incubation of the biotinylated secondary antibody (45’, 1/300, DAG-B, Santa Cruz SC2042) is followed by signal amplification (30’, Streptavidin-Alkaline Phosphatase, Abcam 64268) and visualization using green chromogen (Enzo ADI-950-160-1). Slides are mounted with Faramount (DAKO, S3025). Every section was divided in four quadrants, each of which was scored (1: no osteopontin, (Fig 2A) to 5: maximal osteopontin (Fig 2B)) for osteopontin presence. Quadrant scores were averaged in a blinded manner and used for analysis of SMC phenotype. Negative control stainings (no primary antibody, data not shown) were thoroughly studied before, to avoid taking blood remains into account in our scoring system.

After animal sacrifice by cervical dislocation, tumors and multiple organs were resected and embedded in paraffin. Four micrometer sections were generated followed by immunohistochemistry: Sections were fixed with 4% paraformaldehyde (Sigma), washed, and stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) indicative of apoptotic cells (life technologies, USA), and 4′,6-diamidino-2-phenylindole (DAPI) for labelling cell nuclei. The depth of tissue penetration in tumor nodes was determined by detecting the deepest apoptosis cluster (n = 14 mice with five measurements per tumor node). Proliferating cells were stained with anti-mouse Ki-67 (IHC00375; Bethyl, USA) and HRP labelled anti-rabbit antibodies (K4002; Dako, USA), and DAB substrate (Dako) was added before hematoxylin staining and mounting of samples on microscopy slides using Faramount (Dako). For determination of the cell proliferation index, Ki-67 positive cells and Ki-67 negative cells were counted manually. Tissue sections were microscopically investigated using a Keyence BZ-9000 microscope and evaluated with BZ-II-Analyzer 4.6.2.2 software (Keyence, Japan).

After preliminary testing, Gap27 connexin mimetic peptide (G1794; Sigma-Aldrich) mimicking the aa204–214 sequence on the 2nd extracellular loop of Cx43 (SRPTEKTIFII) was added to the culture media in 200 μM final concentration onto each coverslip cultures containing 2–4 × 10⁵ cells isolated from reactive human tonsils. Cell phenotype, all cell numbers, and average cell numbers within clusters were monitored and compared between Gap27 treated cultures and cultures treated with either 100 µg BSA (untreated) or a scrambled peptide sequence (TFEPDRISITK) at 2 , 4, 6, 12, 16, and 24 h in at least 3 parallel coverslip cultures each time by counting 300 cells on each coverslip. Coverslip cultures were either immunostained (see below) or analyzed after nuclear staining with 7-aminoactinomycin D (7-AAD; 1 : 1000; Invitrogen, Carlsbad, CA) for 1 min, postfixed in 4% neutral buffered formaldehyde for 10 min, and mounted using Faramount (Dako, Glostrup, Denmark). Samples were tested with Leica TCS4D confocal laser scanning microscope (Leica Lasertechnique, Heidelberg, Germany) using single and multichannel fluorescence combined with differential interference (Nomarski) optics.