By Steven M. Powell
The contents of Colorectal melanoma: equipment and Protocols target to show investigators in the entire key genetic, mobile, and molecular organic tools of studying colorectal tumors. The concentrated innovations and assays are defined in enough element to permit researchers to begin an scan on colon tumors and continue from commencing to finish as though the professional within the box who has played those reports have been guiding them on the bench. Of notice, many of the chapters during this quantity are written through these scientists who p- neered those equipment and assays of their respective fields. The chapters in Colorectal melanoma: tools and Protocols describe “state of the artwork” the way to research colorectal tumors, starting from gross mic- dissection of specimens to express molecular analyses. integrated are coverages of mutational assays, instability trying out, immunohistochemical assays, chro- somal reviews, and gene expression analyses. The objective of our quantity is to facilitate the functionality of colorectal tumor organic experiments through investigators at numerous degrees of training―from graduate scholars and postdoctoral fellows to central investigators who wish to enhance our figuring out of colon melanoma improvement.
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Extra resources for Colorectal Cancer: Methods and Protocols (Methods in Molecular Medicine)
2. 44. 81. 8. 2. 3. Equipment (see Note 3 and Fig. 1) 1. 2. 3. 4. 5. 7. Fine tip transfer pipet (sterile). Centrifuge tubes 50 mL. One plastic surgical cutting board 12" × 12". Forceps (small) (sterile). Surgical scissors (sterile). Flat bottom plastic disposable containers with tops. 3. 1. Obtaining the Specimen Obtain the surgical specimen. Select an appropriate area based on clinical diagnosis. Place tissue in 100 mL of ice-cold HBSS in a plastic disposable Epithelial Cell Isolation from Colon 17 Fig.
Probe preparation (labeling, precipitation). Denaturation. Hybridization. Washing. Detection. Microscope analysis. The most crucial part for the successful FISH experiment is a quality of slide preparation. In our experiments fresh tumor tissue was found to be the FISH Techniques to Study Cancer 39 best for touch preps as well as frozen tissue samples. Both provide well separated nicely-shaped interphase cells without multilayer tissue fragments. Frozen tissue sections usually give less satisfying results.
Dissolving the DNA may require 1–2 d incubation with intermittent mixing. 9. 4% agarose gel electrophoresis to estimate the DNA fragments’ length. 1. 2. 3. 4. 5. 6. 7. 8. 2. Preparation of Metaphase Slides (see Note 2) 1. Adjust the humidity to 70–80% and the temperature to 25°C in a hood. 2. From a distance of 30 cm, drop one to two drops from your fixed cell suspension onto the slide. 3. Keep the slide 5 min inside the humid hood before taking it out. 4. Check your metaphases with a phase-contrast microscope and accept only slides with black spread metaphases.
Colorectal Cancer: Methods and Protocols (Methods in Molecular Medicine) by Steven M. Powell