Computational protocol: Activin receptor-like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes

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Protocol publication

[…] Tumours were harvested and fixed in 10% neutral-buffered formalin. Fixed samples were then dehydrated with 70% ethanol, embedded in paraffin and sectioned at 3 μm. For immunohistochemistry, sections were stained with anti-CD8, anti-Eomes, anti-phospho-Smad3 (Abcam), anti-phospho-Smad2 (Cell Signaling Technology) and anti-Smad4 (Santa Cruz) antibodies in antibody diluent overnight and stained with HRP/DAB method (Dako). Sections were then counterstained with haematoxylin. Slides were observed using an optical microscope, Imager Z1 (Carl Zeiss). Expression of Smad4 in B16 melanomas was quantified using ImageJ software (Image Processing and Analysis in Java, National Institutes of Health, USA). [...] Freshly isolated spleen, dLN cells from melanoma-bearing mice were fixed on the slides by 3.7% formaldehyde. PLA was performed using the Duolink II Fluorescence kit (OLINK) according to the manufacturer's protocol using the rabbit antibodies against: Smad2, Smad3, phospho-Smad2, phospho-Smad3, Smad4 and mouse antibodies against: Smad2/3 and ubiquitin (Cell Signaling Technology, BD Bioscience). In order to detect and quantify endogenous Smad protein expression, single recognitions were performed for each Smad protein by using a combination of target specific rabbit primary antibody and its respective secondary antibodies conjugated with oligonucleotides (PLA probe anti-rabbit PLUS and PLA probe anti-rabbit MINUS). In order to detect close proximity of two proteins (<40 nm), double recognitions were performed by using a combination of two target specific primary antibodies raised in different species (rabbit anti-Smad2 and mouse anti-ubiquitin, rabbit anti-Smad3 and mouse anti-ubiquitin, rabbit anti-Smad4 and mouse anti-ubiquitin, mouse anti-Smad2/3 and rabbit anti-Smad4) and their respective secondary antibodies conjugated with oligonucleotides (PLA probe anti-rabbit PLUS and PLA probe anti-mouse MINUS). Technical negative controls using one primary antibody for each combination of double recognitions showed no background signals. After incubation of the slides with blocking solution for 30 min at 37°C, they were incubated with primary antibodies diluted in the antibody diluent overnight at 4°C, in PLA probe solution for 1 h at 37°C and in ligation-ligase solution for 30 min at 37°C with washing with wash buffer A in the interim of each step. The slides were incubated in amplification-polymerase solution for 100 min at 37°C and then washed in wash buffer B. To co-stain CD8, rat anti-CD8 antibody was added in the antibody diluent with primary antibodies for PLA and the slide were incubated with Alexa Fluor 488 conjugated anti-rat IgG (Abcam) after washing in wash buffer B. Nucleus was stained with DAPI. Then, the slides were dried at room temperature in the dark. Slides were observed using a confocal microscope, LSM700 (Carl Zeiss). PLA signals were quantified using BlobFinder software (Centre for Image Analysis, Uppsala University). [...] Statistical analyses were performed using analysis tools provided on the VassarStats statistical computation site (http://vassarstats.net/). Data were analysed using the two-tailed unpaired Student's t-test and two-way ANOVA test. A p-value <0.05 was considered to indicate statistical significance. […]

Pipeline specifications

Software tools ImageJ, BlobFinder, VassarStats
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Melanoma, Neoplasms