Cy3 conjugated goat anti mouse antibody

To label GAD-67 immunoreactive neurons, a 1-in-5 series of free-floating sections was collected from the cryoprotectant solution, washed three times for 5 min each in 0.1 M PBS, and incubated in a blocking solution containing 10% normal goat serum (Millipore Bioscience Research Reagents, Temecula, CA) and 0.5% Triton X-100 in PBS for 1 hour at room temperature. The sections were then incubated with mouse anti-GAD-67 serum (1∶1000, MAB5406; Millipore Bioscience Research Reagents), 5% normal goat serum, 0.3% Triton X-100, and 1% bovine serum albumin overnight at 4°C. After rinsing three times for 10 min each in 0.1% Triton X-100 in PBS, the sections were incubated with Cy3-conjugated goat anti-mouse antibody (1∶1000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and 0.0001% 4,6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich) in PBS for 1 hour at room temperature. After a final rinse in PBS for 10 min, sections were mounted on slides, air-dried for at least 30 min, and coverslipped with ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA).

To determine the subcellular localization of the product of the MSX1 minigene with the intronic mutation, COS7 cells were transfected with the minigene or cDNA expression plasmids for the products using Lipofectamine 2000 (Invitrogen) as described previously [23 (link)]. Forty-eight hours post-transfection, the cells were fixed with 4% paraformaldehyde/Tris-buffered saline (TBS) and permeabilized with 1% Triton X-100/TBS prior to incubation with an anti-FLAG M2 monoclonal antibody (1:2000). The cells were then incubated with DAPI (1 mg/ml) and Cy3-conjugated goat anti-mouse antibody (1:1000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) in phosphate-buffered saline (PBS). After washing the cells with PBS three times, immunostaining signals were visualized under an Olympus BH-2 microscope.

For detection of hypoxia, mice were treated with 60 mg/kg of pimonidazole HCl intraperitoneally (Hypoxyprobe-1 kit; Hypoxyprobe) 1 h prior to euthanasia. Colon samples were fixed in 10% phosphate-buffered formalin, and paraffin-embedded tissue was blocked with mouse-on-mouse blocking reagent (Vector Labs) and probed with mouse anti-pimonidazole monoclonal IgG1 (monoclonal antibody 4.3.11.3). Then, slides were stained with Cy3-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories). Samples were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) using SlowFade Gold mountant. Samples were scored based on the degree of colonic epithelial hypoxia (0, no hypoxia; 1, mild focal hypoxia; 2, moderate multifocal hypoxia; 3, intense diffuse hypoxia). Representative images were obtained using a Zeiss Axiovert 200 M fluorescence microscope and were brightness adjusted.

HUVEC were seeded onto glass coverslips and treated with CPA as described above. Eight days after CPA-treatment, the cells were fixed in 4% paraformaldehyde in PBS (Morphisto GmbH, Offenbach am Main, Germany) for 10 min, permeabilized in 0.25% Triton X-100 (Carl Roth GmbH+Co. KG, Karlsruhe, Germany) for 3 min and blocked in 1 × phosphate-buffered saline (PBS, Bio&Sell GmbH, Feucht, Germany) with 3% bovine serum albumin (BSA, Carl Roth GmbH) for 30 min. Thereafter, cells were first incubated with anti-phospho-histone H2AX mouse monoclonal IgG (Merck Millipore) diluted 1:1000 in 1 × PBS/1% BSA for 1 h at room temperature. Then the cells were incubated with polyclonal Cy3-conjugated goat-anti-mouse antibody (Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) and 0.2 μg/mL DAPI (Carl Roth GmbH) diluted in 1 × PBS/1% BSA for 1 h at room temperature. Immunofluorescence images were obtained with a ZEISS LSM 800 (Carl Zeiss, Jena, Germany). The cell nuclei as well as γH2AX foci were counted using the bioimage analysis software QuPath.

Cells were grown to mid-log phase in YEPD rich media at 30°C and washed in spheroplasting solution (1.2 M sorbitol, 0.1 M potassium phosphate, 0.5 M MgCl₂, pH 7) and digested in spheroplasting solution with 10 mM DTT and 150 µg/mL Zymolase 20T at 37°C for 20 minutes similar to previously described ([72] (link)). The digestion was halted by addition of ice-cold stop solution (0.1 M MES, 1 M sorbital, 1 mM EDTA, 0.5 mM MgCl₂, pH 6.4) and spheroplasts were lysed with 1% vol/vol Lipsol and fixed on slides using 4% wt/vol paraformaldehyde/3.4% wt/vol sucrose ([73] (link)). Chromosome spread slides were incubated with the mouse monoclonal antibody S9.6 (1 µg/mL in blocking buffer of 5% BSA, 0.2% milk and 1× PBS). The slides were further incubated with a secondary Cy3-conjugated goat anti-mouse antibody (Jackson Laboratories, #115-165-003, diluted 1∶1000 in blocking buffer). For each replicate, at least 100 nuclei were visualized and manually counted to obtain the fraction with detectable DNA:RNA hybrids. Each mutant was assayed in triplicate. Mutants were compared to wild type by the Fisher's exact test. To correct for multiple hypothesis testing, we implemented a cut off of p<0.01 divided by the total number of mutants compared to wild type, meaning mutants with p<0.00024 were considered significantly different from wild type.

Circulating hemocytes were collected from wandering third-instar larvae. Cells were attached to slides at 29°C for 60 min. Cells were then fixed for 10 min in 4% paraformaldehyde in PBS, blocked with 0.5% Triton X-100 plus 5% bovine serum albumin (in 1× PBS) for 1 h, incubated with primary antibodies anti-P1 (a kind gift from Dr I. Ando, Biological Research Center, Szeged, Hungary; 1:1000), anti-4D4 (Developmental Studies Hybridoma Bank; 1:1000) or anti-Lz (Developmental Studies Hybridoma Bank; 1:1000) at 4°C overnight. The cells were then washed three times with 1× PBS, incubated with Cy3-conjugated goat anti-mouse antibody (Jackson ImmunoResearch; 1:2000), washed three times with 1× PBS, mounted and imaged using a Zeiss Axio Imager with ApoTome2.