To fix samples/cells, slides/coverslips were fixed and permeabilised for 15 min in dry methanol at −20 °C and rehydrated in PBS. Samples/cells were blocked (donkey serum 1:30) for 1 h at 37 °C in a humified chamber incubated with primary antibody (either Anti-pSer473AKT1 or TRIB2) for 1 h at 37 °C in a humified chamber followed by PBS washing and incubation with a fluorescent secondary antibody (Molecular Probes A21207 or A11055) for 1 h at 37 °C in a humified chamber. Antibody solutions were made in PBS (Sigma). Following labelling procedures, samples/cells were mounted on glass slides in Clear Mount mounting solution (Invitrogen).
Clear mount mounting solution
Manufactured by Thermo Fisher Scientific
Clear Mount mounting solution is a water-based, non-toxic mounting medium designed for microscopy applications. It provides a clear, permanent seal for mounting specimens on microscope slides.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti fluorescence microscope, by Nikon (2 mentions)
Hematoxylin, by Merck Group (2 mentions)
A21207, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Immpact novared peroxidase substrate kit, by Vector Laboratories (1 mentions)
Bx61 microscope, by Olympus (1 mentions)
Optimal cutting temperature compound, by Sakura Finetek (1 mentions)
Oil red o stock solution, by Merck Group (1 mentions)
Slowfade gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Insulin syringe, by BD (1 mentions)
Vwr micro slides, by Avantor (1 mentions)
6 protocols using clear mount mounting solution
1
Immunofluorescence Staining of Cells
2
Sequential Immunohistochemistry for EMT Markers
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Sequential IHC was performed with 4 μm of FFPE tissue sections. Following deparaffinization, sections were rehydrated and stained with hematoxylin (S3301, Dako) for 1 min, cover-slipped with a ClearMount™ Mounting Solution (008010, Invitrogen). Tissue scan was proceeded using an Aperio ImageScope (Leica Biosystems). Slides were de-coverslipped in water for 1 h and subjected to endogenous peroxidase blocking followed by heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 15 min. Sequential IHC (staining, scanning and antibody stripping) was performed according to a protocol on previous report56 (link),57 (link). Slides were blocked with 4% skim milk in TBST for 30 min and incubated in primary antibodies: anti-Twist1 (ab50887, 1:200), anti-Prrx1 (HPA051084, 1:200), and anti-TNC (ab108930, 1:500) for 60 min at room temperature. Secondary antibody that was required to be compatible with the primary host was incubated for 1 h, and protein was subsequently detected using the DAKO-Envision Dual Link Labelled Polymer (Anti-Rabbit) (#K5007, Dako Botany, NSW, Australia) for 30 min and the ImmPACT NovaRed Peroxidase Substrate Kit (#SK-4805, Vector Laboratories, Burlingame, CA, USA) for 3–4 min at room temperature.
3
Oil-Red O Staining of Frozen Liver Sections
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Fresh liver tissue was frozen in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and cryostat sections (7 µm-thick) were fixed in ice-cold 10% formalin for 10 min. Fresh oil-red O solution was prepared by dilution of the oil-red O stock solution (Sigma, 3.5 g/l in 99.5% isopropyl alcohol) in distilled water to a final concentration of 0.2%. This solution was mixed thoroughly and filtered through a 0.2-µm filter before use. Next, slides were placed in absolute isopropyl alcohol for 2 min. Subsequently, the sections were stained in pre-warmed oil-red O solution for 20 min in an oven at 60°C, rinsed in distilled water and counterstained with haematoxylin, followed by washing with running tap water. The slides were mounted with Clear Mount™ Mounting solution (Invitrogen, Eugene) and examined using an Olympus BX61 microscope.
4
Immunohistochemical Analysis of Immune Cells
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Formalin-fixed, paraffin-embedded lung and liver tissue sections were stained with hematoxylin and eosin (H&E; Sigma Chemical, St Louis, MO). Frozen tumor sections were fixed with cold acetone, acetone plus chloroform (1:1), and acetone. Tissue sections were blocked with blocking buffer (5% normal horse serum and 1% normal goat serum in phosphate-buffered saline solution) and incubated with rat anti-mouse CD8α antibody (clone YTS105.18; AbD Serotec, Raleigh, NC). For FOXP3 staining, sections were blocked with blocking buffer containing 0.1% Triton X-100 for 40 min and incubated with rat anti-FOXP3 antibody (#12653; Cell Signaling Technology, Danvers, MA) overnight at 4 °C. The next day, tissue sections were blocked and incubated with goat anti-rat horseradish peroxidase secondary antibody (Life Technologies) for 1 h at room temperature, which was followed by DAB staining for 5~10 min at room temperature. Nuclei were then counterstained with hematoxylin (Sigma Chemical), and tumor sections were mounted with ClearMount Mounting Solution (Life Technologies). Slides were visualized under a Nikon eclipse Ti fluorescence microscope (Nikon Instruments, Melville, NY).
5
Immunohistochemistry and Immunofluorescence of Frozen Tumor Sections
6
Measuring Drosophila Wing Length
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To measure wing length, the right wing of each specimen was removed by cutting near the tegula using the tip of a small metal syringe (8 mm BD 3/10 ml/cc Insulin Syringe, Becton, Dickinson and Company, Franklin Lakes, NJ). Each wing was placed flat on a glass slide (VWR Micro Slides, Cat. No. 48300-037, VWR International LLC, Radnor, PA). All flies were stored in 80% ethanol prior to and during wing removal. Up to 10 female and 10 male wings on slides were arranged and fixed to the slide using a protein fixative (ClearMount Mounting Solution, LifeTechnologies, Frederick, MD) and slide cover (VWR Plastic Cover Slip, Cat. No. 48376-049, VWR International LLC). Additionally, the whole abdomen melanization of each specimen was recorded as ‘light’, ‘medium’, or ‘dark’. This was a subjective assessment and lab-reared summer morphs were used as the baseline ‘light’ category. All slides were then photographed at identical magnification parameters (Leica S6D and Microscope Camera MC120 HD, using the Leica Application Suite V 4.6.0, Leica Microsystems Inc., Buffalo Grove, IL) and measured using ImageJ (imagej.nih.gov/ij/). Two length measurements were made for each wing along the IV vein, as described in Shearer et al. (2016) (link). For analysis, these two lengths were summed and considered as total wing length.
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