Images were acquired using a fluorescent stereoscope (Leica) equipped with a K5 camera (Leica) with resolution (h x v) 2048 × 2048 pixels and pixel size (h x v) 6.5 × 6.5 μm, at an image magnification of 100x with no binning. Images were obtained right after implantation (0 days post injection, dpi) and after 72 h incubation (3 dpi) and analyzed by using the HuginMunin software v2.7.0.0 (BioReperia AB, Linköping, Sweden). Tumor sizes were analyzed as the area of labeled tumor cells at 0 dpi and 3 dpi, and the number of disseminated cells to the caudal venous plexus were manually counted. Relative tumor size was calculated by dividing the tumor area at 3 dpi by the area at 0 dpi in the same embryo and multiply by 100.
Fluorescent stereoscope
Manufactured by Leica
Sourced in Germany
The Leica Fluorescent Stereoscope is an optical microscope designed for the observation and analysis of fluorescent-labeled specimens. It provides a stereoscopic, three-dimensional view of the sample, allowing for detailed examination and documentation.
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Lab products found in correlation
4 protocols using fluorescent stereoscope
1
Quantifying Tumor Growth and Metastasis
2
Hypothalamic Microdissection for RNA Profiling
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All mice were sacrificed at ZT 5. Brains were immediately extracted and dropped into ice cold Hanks’ Balanced Salt Solution (HBSS). After 2 minutes, brains were embedded in low melting point agarose (Precisionary Instruments, Natick, MA) and sectioned at 400 μm on a Compresstome VF-200 Vibrating Microtome (Precisionary Instruments, Natick, MA, USA) into DNAse/RNAse free 1x PBS. Hypothalamic sections of interest were immediately collected into RNAprotect (Qiagen, Hilden, Germany) and kept in RNAprotect at 4°C overnight. The next day Lepr-Cre; TdTomato positive cells were visualized using a fluorescent stereoscope (Leica, Wetzlar, Germany). tdTomato fluorescence was used to approximate DMH and SCN boundaries during microdissection of these regions. DMH and SCN microdissected tissue samples were placed into Eppendorf tubes separated by brain region and feeding condition, and stored in −80°C until nuclei isolation.
3
Brain Dissection for Single-Cell Analysis
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All mice were euthanized at ZT5. Brains were immediately extracted and dropped into ice-cold Hanks’ balanced salt solution. After 2 min, brains were embedded in low melting point agarose (Precisionary Instruments, Natick, MA) and sectioned at 400 μm on a Compresstome VF-200 Vibrating Microtome (Precisionary Instruments, Natick, MA, USA) into deoxyribonuclease/ribonuclease-free 1× phosphate-buffered saline (PBS). Hypothalamic sections of interest were immediately collected into RNAprotect (QIAGEN, Hilden, Germany) and kept in RNAprotect at 4°C overnight. The next day Lepr-Cre;TdTomato–positive cells were visualized using a fluorescent stereoscope (Leica, Wetzlar, Germany). tdTomato fluorescence was used to approximate DMH and SCN boundaries during microdissection of these regions. DMH and SCN microdissected tissue samples were placed into Eppendorf tubes separated by brain region and feeding condition and stored in −80°C until nucleus isolation.
4
Xenopus Cytosol Microtubule Depolymerization Assay
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Interphase cytosol extracts from Xenopus eggs were obtained as described previously (Allan and Vale, 1991 (link)). MT depolymerisation was assessed in a microscopic flow chamber (Vale and Toyoshima, 1988 (link)) where Xenopus cytosol (1 µl cytosol diluted with 20 µl acetate buffer) was incubated for 20 min to allow MTs to polymerise. Then cytosol was exchanged by flow through with Efa6-ΔCterm::GFP, MCAK or synthetic MTED peptide (all 20 nM in acetate buffer pH 7.4: 100 mM K-Acetate, 3 mM Mg-Acetate, 5 mM EGTA, 10 mM HEPES), and MT length changes observed by recording 10 random fields via VE-DIC microscopy (Allan, 1993 (link); Allan and Vale, 1991 (link)). MT polymerisation was analysed in a microscope flow cell containing 9 µl diluted Xenopus egg cytosol (see above) to which 1 µl acetate buffer was added, either alone or containing 20 nM MTED. After 10 min, 20 random fields were recorded via VE-DIC microscopy for each condition and the numbers of MTs per field counted.
For the in vivo assay, Xenopus embryos were injected in one blastomere at the 4 cell stage with 200 ng of mRNA encoding Efa6-Nterm::GFP or mCherry alone. The embryos were imaged at stage 10.25 (Heasman, 2006 (link)) with a Leica fluorescent stereoscope.
For the in vivo assay, Xenopus embryos were injected in one blastomere at the 4 cell stage with 200 ng of mRNA encoding Efa6-Nterm::GFP or mCherry alone. The embryos were imaged at stage 10.25 (Heasman, 2006 (link)) with a Leica fluorescent stereoscope.
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