Mowiol mounting medium

Coverslips in 24-well plates were treated with Collagen I (rat tail, 50 µg/ml, Gibco) for 1 h at room temperature. After 3 PBS washes, HEK293T cells were cultured overnight, followed by transfection of 250 ng of FAME-pEGFP-N1 using Lipofectamine LTX® and Plus™ Reagent (Invitrogen). After overnight incubation, the cells were fixed using 4% paraformaldehyde in PBS for 20 min at room temperature. After 3 PBS washes, cells were either directly stained with primary anti-1700011H14Rik/FAME antibody (Santa Cruz) and subsequent donkey anti-mouse 555 secondary antibody (Invitrogen), or treated with 1x Dako Target Retrieval Solution, pH 9 (Agient) for 40 min in a steamer at 97 °C. Next, cells were washed 3 times with PBS-T and stained sequentially with primary anti-FAME and anti-GFP (Abcam, ab13970) diluted in a mixture of 5% donkey serum, 20% dimethyl sulfoxide (DMSO) (Sigma) and 75% PBS for one hour, followed by 3 PBS-T washes. Secondary antibody staining with Donkey anti-mouse 555 (Alexa Fluor™) and donkey anti-chicken 647 (Invitrogen) was done for 1 h. Following 3 PBS-T washes, cells were stained with 4’,6-Diamidino-2-phenylindole (DAPI) at a 1:10.000 dilution and mounted with Mowiol mounting medium (Sigma). Fluorescence images were obtained by Leica DMi8 automated fluorescence microscopy.

Transmission electron microscopy (TEM) was performed as described previously (65 (link)), with the exception that grids were placed on a drop of bacterial suspension at 22°C for 10 min and the samples were stained with an aqueous 0.5% ammonium molybdate solution for 10 min before being viewed in a Philips CM100 transmission electron microscope. Immunofluorescence (IF) microscopy was performed as described previously (48 (link)), except that the P. aeruginosa strains were grown to an OD₆₀₀ of 0.2 prior to incubating the bacteria on poly-l-lysine-coated glass coverslips. P. aeruginosa pilin-specific antiserum (gift from E. C. Gotschlich, Rockefeller University) was used as a primary antibody for Tfp labeling, followed by an Alexa Red 594-conjugated goat anti-rabbit IgG (Molecular Probes). P. aeruginosa cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) at 1 μg/mL in Mowiol mounting medium (Sigma) containing 2% 1,4-diazabicyclo(2)octane (DABCO) prior to viewing with a Nikon Eclipse C400 fluorescence microscope.

Whole-mount in situ hybridization (WISH) was performed as previously described (Pearson et al., 2009; (link)
Sasidharan et al., 2013; (link)
King and Newmark, 2013 (link)). miRNA-based WISH was carried out using digoxigenin-labeled miRCURY LNA probes obtained from Exiqon. For immunostaining, animals were fixed using Carnoy's solution as described (Sánchez Alvarado and Newmark, 1999 (link)). Rabbit anti-ARRESTIN (1:5000, clone VC-1; gift of Dr Kiyokazu Agata, Kyoto University, Japan) and mouse anti-SYNORF-1 (1:100, DHSB) primary antibodies were used. Species-specific secondary antibodies were obtained from Molecular Probes (1:400). Hoechst 33342 (25 µg/ml; Sigma) was used as a nuclear counterstain. Animals were mounted with Mowiol mounting medium (Sigma) and stored at 4°C until imaging.

Mel270 cells were seeded in 8-chamber polystyrene-vessel tissue-culture glass slides (Corning, Kennebunk, ME, USA) at 3000 cells per chamber and allowed to attach for 24 hours. Cells were incubated with 300 pM AU-011 for 4 hours at 4°C or 37°C. Slides were washed with PBS and incubated with a cell surface marker (CD44-FITC; Thermofisher) at 4°C for 30 minutes. Then, the slides were washed 3 times with PBS and fixed with 1% formalin (J.T. Baker) for 10 minutes at room temperature. The cells were washed again with PBS and stained with DAPI (4',6-diamidino-2-phenylindole, Sigma, St. Louis, MO, USA) at 5 µg/mL for 5 minutes. Slides were again washed 3 times with PBS, after which coverslips were mounted on the glass slides using Mowiol mounting medium (Sigma) supplemented with 2.5% DABCO and sealed with nail polish. Slides were imaged on a Leica SP8 fluorescence microscope.

To detect apoptotic cells in melanoma tissue, fluorescent TUNEL staining (In Situ Cell Death Detection kit, fluorescein; Roche Applied-Science) was performed according to the manufacturer’s protocol. Briefly, after deparaffinization and rehydration, slides were incubated with proteinase K solution for 30 min at 37 °C and washed in PBS for 2 min, and after blocking, incubated with TUNEL reaction mixture for 1 h at 37 °C. After washing, the slides were counterstained with PI, washed, and mounted with Mowiol mounting medium (Sigma Aldrich Chemie, St. Louis, MO, USA) in order to be analyzed with SP5 confocal microscope (Leica Microsystems).

To visualize biocytin filling, slices were incubated in streptavidin-Alexa Fluor 488 conjugate (Invitrogen; concentration, 1:200) with 0.3% Triton X-100 in 0.01 m PBS at room temperature for 3 h in dark. After washing three times with 0.01 m PBS for 30 min each, sections were mounted in glass slides (Menzel–Glazer), coverslipped (Marienfeld) with Mowiol mounting medium (Sigma-Aldrich) and the edges sealed with clear nail polish. For tissue collection, VGat-Cre-tdtomato mice were deeply anesthetized with a lethal amount of a mixture of 12% of ketamine (Imalgene 1000, Merial) and 8% of xylazine (Rompun 2%, Bayer) in saline solution and perfused transcardially with 0.01 m PBS followed by 4% PFA in PBS. The brains were removed, fixated in the 4% PFA solution for ∼1 h and transferred to a 30% sucrose (Sigma-Aldrich) in 0.01 m phosphate buffer and 0.1% sodium azide (ACROS Organics) to allow cryopreservation. Brains were then frozen and coronal sections with 45-μm thickness were obtained from the VMH using a freezing sliding microtome (SM2000R, Leica).
Histologic sections and fixed ex vivo brain slices were imaged with a Zeiss LSM 710 confocal laser scanning microscope with a 10× and a 25× magnification objectives.

Apoptotic index was measured as previously described [23 (link)] using a propidium iodide (PI) (Sigma-Merck) staining solution containing 5 μg/mL PI, 0.1% Triton X-100, 0.1 M EDTA and 25 U/mL RNAse (Sigma) (20 min incubation at 37 °C) in cells fixed with methanol:acetic acid (3:1) for 30 min at RT. Cells were washed and coverslipped using Mowiol mounting medium (Sigma). Cells undergoing apoptosis were scored under an inverted fluorescence microscope (Eclipse TE300, Nikon, Izasa Scientific, Alcobendas, Madrid, Spain) at high magnification (x60) following standard morphological criteria, that is, visualization of nuclear condensation, shrinkage and fragmentation. Quantification was performed in a blinded manner as follows: 10–20 microscopic fields per plate were randomly chosen throughout the entire plate in order to reach a minimum of 1000 cells per treatment. Both apoptotic and non-apoptotic nuclei were counted in each field to obtain the percentage of apoptosis/field. The score for each plate corresponded to the mean percentage of all fields. Each condition was run in triplicates, so the final score per condition was the mean percentage of three plates.