Ds qi2 monochrome camera

Chromosome spreads for immunofluorescence analysis were prepared as described previously (21 (link),46 (link),47 (link)). Briefly, cells were lysed and fixated onto a clean slide using 1% Lipsol and 3% paraformaldehyde containing 3.4% sucrose. Then, the slides were soaked in 0.2% Photo-Flo (Kodak, 146–4510), transferred to Tris-Buffered Saline (TBS) (136 mM NaCl, 3 mM KCl and 25 mM Tris–Cl, pH 8.0) and incubated for 15 min. For immunostaining, the following antibodies were used in this study: rabbit polyclonal Zip1 antibody (diluted 1:200; Santa Cruz Biotechnology, sc-33733); mouse monoclonal GFP antibody (diluted 1:5000; Santa Cruz Biotechnology, sc-9996); primary mouse monoclonal Myc antibody (diluted 1:200; Santa Cruz Biotechnology, sc-40); primary rabbit polyclonal HA antibody (diluted 1:200; Santa Cruz Biotechnology, sc-805); secondary TRITC-conjugated goat anti-rabbit lgG (diluted 1:300; Jackson ImmunoResearch, 111–025-003); secondary Alexa 488-conjugated goat anti-mouse IgG (diluted 1:300; Jackson ImmunoResearch, 115–545-003). Images were acquired using a Nikon Eclipse Ti fluorescence microscope equipped with a Nikon DS-Qi2 monochrome camera. Image deconvolution was adjusted with Nikon NIS software.

Immunofluorescent staining of human lung cancer tissue sections was performed as described elsewhere^{54 (link)}. Bright field and fluorescence Images were acquired with a 20X (0.75 NA) objective on a Nikon A1+ confocal or with a 10X (0.45 NA) objective on a Nikon Eclipse Ti inverted widefield microscope (Nikon Corporation, Tokyo, Japan) equipped with DS-Qi2 monochrome camera (fluorescence) and DS-Ri2 color camera (color Brightfield) using NIS Elements software (Nikon). Raw images were deconvolved with Huygens Professional version 15.05 (Scientific Volume Imaging, The Netherlands, https://svi.nl/) and processed in ImageJ (NIH, Bethesda, https://imagej.nih.gov/ij). Briefly, deconvolved images were thresholded, smoothened and watershed segmented before creating a mask for DAPI channel to score for total number of cells per field. Other channels were processed identically and multiplied with the nuclear mask before scoring for number of single positive cells. Double positive cells were identified using RG2B colocalization plugin. Scores were expressed as percentage.

For analysis of focal adhesion dynamics, 7 × 10⁴ cells were seeded on collagen-coated glass-bottom plates 48 h post transfection of a paxillin-GFP construct. Transfected cells were incubated in serum-free RPMI1640 medium for 16 h and stimulated with 10% FBS. Cells were imaged using an Eclipse Ti2 microscope (Nikon) equipped with a DS-Qi2 monochrome camera (Nikon). Images were captured every minute for 1 h using a Plan Fluor 10×/0.30 objective lens (Nikon). Focal adhesion assembly was measured using NIS-Elements AR 5.20.00 software.

Approximately 75 L4 animals were picked to new plates, harvested 24 hours later as day 1 adults in S buffer, washed, fixed with 60% isopropanol, and stained for 7 hours with 0.3% Oil Red O as previously described [16 (link)]. Animals were mounted on 2% agarose pads and imaged with a Nikon SMZ-18 Stereo microscope equipped with a DS-Qi2 monochrome camera for intensity analyses or a Nikon Ti2 widefield microscope equipped with a DS-Fi3 for representative color images. Quantification of Oil Red O staining was performed in ImageJ by manually outlining worms and determining the mean gray value in the worm area. Each value was subtracted from 65,536, the maximum gray value for 16-bit images. The data were plotted using Prism 9 and a one-way ANOVA with a Bonferroni correction for multiple testing was performed to calculate P values.

IHC of tumor sections was performed by Sophistolab (Muttenz, Switzerland) on a Lecia BondMax instrument using Refine HRP-Kits (Leica DS9800). All buffer-solutions were purchased from Lecia Microsystems Newcastle, Ltd and used according to the manufacturer’s guidelines. Paraffin-slides were de-waxed, pre-treated and incubated as follows: ER-solution 2 for 10 min at 95 °C, ER-solution 2 for 20 min at 100 °C and ER-solution 2 for 30 min at 100 °C. pFRS2; Rabbit anti-Phospho FRS2 (PhosphoY 436, Abcam Limited; ab193363), dilution 1:150; phospho-p44/42 MAPK (Erk1/2_{Thr202/Tyr204}) Rabbit mAb (Cell Signaling Technology #4370), dilution 1:1600. The IHC images were captured digitally using a Nikon Epifluorescence Eclipse Ti2 equipped with a Nikon DS-Ri2 color and a Nikon DS-Qi2 monochrome camera.

Fluorescent labeled slides were examined using the Eclipse E400 microscope (Nikon Inc.; Melville, NY, USA). Pictures were taken with DS-Qi2 Monochrome Camera (Nikon Inc.; Melville, NY, USA) and the computer Imaging Software NIS-Elements A (Nikon Inc.; Melville, NY, USA). A total of five pictures of aleatory picked fluorescently labeled areas at a magnification of 20X were captured per slide. Aleatory areas of tissue were selected to capture the pictures based on similar cell ratios as determined by the number of nuclei. A total of four pictures were captured with different filters in the same area, one for each fluorescent label secondary antibody and DAPI using one second exposure. The wavelengths to excite each fluorophore were 358, 488, and 546, and 647 nm. Using the NIS-Elements A software tools of analysis, data were quantified from each picture by counting objects and mean fluorescence intensity of each labeled protein. Data was exported to the software Excel from Office 365® (Microsoft®; Redmond, WA, USA).