Anti mouse cy2

Longitudinal sections of 12–15 μm were collected on glass slides, blocked with PBS with 1% BSA for 1 h at RT and incubated with the following primary antibodies, diluted from 1 : 250 to 1 : 50 in PBS + 1% BSA, ON at 4°C: anti-Pin1 (G8 sc-46660, Santa Cruz), with anti-p63 (4A4 sc-8431, Santa Cruz) and with anti-E-cadherin (36/E 610182, BD) and then incubated with secondary antibodies anti-mouse-Cy2 and anti-rabbit-Cy3 (Jackson ImmunoResearch) diluted 1 : 200, 1 h at RT, washed, stained with DAPI for the nuclei detection and examined with a Zeiss Observer-Z1 fluorescent microscope, equipped with Apotome system. Raw images were digitally processed to normalize the background and optimize the contrast, with Photoshop (Adobe), and mounted with QuarkXpress (Pantone).
Semi-quantitative immunofluorescence analysis was performed with ImageJ-64 (v1.45) software. Images were first converted to grayscale, and the DAPI channel was used to count nuclei. p63 intensity was quantified after background correction and normalized respect to the number of nuclei in the region of interest. Data are presented as mean and s.d. of ∼4/5 different sections of three different embryos. A significant T-test score is indicated by asterisks: *indicates P < 0.05, **indicates P < 0.01.

The day prior to the experiment, 10⁵ U2OS cells were seeded onto 11 mm glass coverslips and allowed to attach overnight at 37 °C/5 % CO₂ in DMEM containing 10 % FBS (Gibco). Cells were treated with 100 μM sodium (meta)arsenite (Sigma Aldrich) for 1 h to induce the formation of stress granules and then with 4 % paraformaldehyde solution at room temperature for 15 minutes followed by blocking and permeabilization with 5 % normal horse serum, 0.1 % digitonin in Tris-buffered saline. Staining was performed with anti-eIF3b (Santa Cruz), anti-SK1-Hedls (Santa Cruz), and patient sera for 1 h at room temperature. Secondary antibodies (anti-goat-Cy3, anti-mouse-Cy2, and anti-human-Cy5) were purchased from Jackson Laboratories and incubated at room temperature for 1 h. Conventional fluorescence microscopy was performed using a microscope (model Elipse E800, Nikon, Tokyo, Japan) with epifluorescence optics with a digital camera (model CCD-SPOT RT; Diagnostic Instruments, Sterling Heights, MI). Images were compiled using Adobe Photoshop software (CS6; Adobe Systems, San Jose, CA).

The experiments were performed in perfusion-fixed (4% paraformaldehyde in PBS) MONs or coronal brain slices in a mixture of 4% paraformaldehyde (#P6148, Millipore Sigma, St-Louis, MO) in PBS and 0.025% glutaraldehyde (#O2957, Fisher Scientific, Pittsburgh, PA). Cryoprotection was achieved in 30% sucrose for 16–18 h. Sections of 50 μm (brains) and 16 μm (MONs) thickness were blocked in 20% normal goat/donkey (50% by volume) serum (#G9663 & G6023, Millipore Sigma, St-Louis, MO) and 1% Triton X-100 (#X100, Millipore Sigma, St-Louis, MO) for 60 min at room temperature. Sections were then incubated in primary antibodies prepared in the same solution and kept overnight at 4°C (see Table 1 for antibodies and dilutions used). After several thorough washes in PBS, the tissue was incubated with secondary antibodies, prepared in 2% normal goat serum for 2 h at room temperature. The secondary antibodies used were donkey anti-rabbit Cy2, anti-mouse Cy2 and anti-rabbit Cy3 and anti-mouse Cy3 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Sections were double or triple labeled to visualize structures of interest.

The experiments were performed in perfusion-fixed (4% paraformaldehyde in PBS) MONs or coronal brain slices in a mixture of 4% paraformaldehyde (#P6148, Millipore Sigma, St-Louis, MO) in PBS and 0.025% glutaraldehyde (#O2957, Fisher Scientific, Pittsburgh, PA). Cryoprotection was achieved in 30% sucrose for 16–18 h. Sections of 50 μm (brains) and 16 μm (MONs) thickness were blocked in 20% normal goat/donkey (50% by volume) serum (#G9663 & G6023, Millipore Sigma, St-Louis, MO) and 1% Triton X-100 (#X100, Millipore Sigma, St-Louis, MO) for 60 min at room temperature. Sections were then incubated in primary antibodies prepared in the same solution and kept overnight at 4°C (see Table 1 for antibodies and dilutions used). After several thorough washes in PBS, the tissue was incubated with secondary antibodies, prepared in 2% normal goat serum for 2 h at room temperature. The secondary antibodies used were donkey anti-rabbit Cy2, anti-mouse Cy2 and anti-rabbit Cy3 and anti-mouse Cy3 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Sections were double or triple labeled to visualize structures of interest.