FISH assay for detection of HER2 amplification in breast cancer
FISH assay for detection of HER2 amplification in breast cancer
Gene amplification is frequently detected in human tumor cells and plays an important role in tumorigenesis. Genome-wide scans of tumor cells using comparative genomic hybridization have revealed gene copy number alterations in many regions of the solid tumor genome. Intensive studies of DNA in altered regions have shown that complex DNA rearrangements in solid tumors involve multiple genes and span several Mb. In chromosomal ploidy changes, gene rearrangements are often accompanied. Although the study of amplified regions of DNA has the potential to identify new genes associated with tumorigenesis, the co-amplification of neighboring genes in these large complex rearrangements is more complex. Many tumors show a large increase in DNA and chromosomal rearrangements, and it has been suggested that the key event in the process of tumorigenesis is genomic instability and that gene amplification is only a consequence.
Operation method
FISH assay for detection of HER2 amplification in breast cancer
Materials and Instruments
Pepsin Neutral formalin buffer Formamide Ethanol Move I. Sections and Preparations 1. Cut thick tissue sections from formalin-fixed paraffin-embedded tissue blocks and spread the sections in a 37°C water bath. 2. attach to positively charged slides. 3. cut another consecutive section of the corresponding tissue and stain it with Hematoxylin and Eosin (H&E) (see Note 2). 1. Confirm the appropriate tumor cell location on the HE section (see Note 3). 2. Check the composition and quality of fixation of the sectioned tissue (see Note 4). 3. Bake the slices in an oven or roaster at 60°C for 2 h (see Note 5). 4. Place the slices in a slicing rack and xylene 3 cylinders for 5 min each to remove paraffin. Pass through anhydrous ethanol 3 times (see Note 6). 5. Dry the slices at 45°C for 3 min in a toaster or on a hot plate. 6. Place the slices in 50 mL of 0.2 mol/LHCl for 20 min at room temperature. 7. Wash the sections in 50 ml of distilled water for 1 min, and wash them twice with 2XSSC for 3 min each time. 8. Place in lmol/LNaSCN at 81°C for 30 min (see Note 7). 9. Wash with distilled water for 1 min and with 2XSSC twice for 3 min each (see Note 8). 10. 3?IOmin at 37°C in pepsin solution (see Note 4 for optimum time). 11. Wash with fresh 50 mL of distilled water for lmin and 50 mL of 2XSSC twice. 12. Dry slices at 45°C for 3 min. 13. Secondary fixation: Place at room temperature in 10% neutral formalin buffer for IOmin (see Note 9). 14. 2XSSC wash 2 times for 3 min each. 15.45°C, 3 min dry slices (see Note]_0). 16. Denature the DNA on the tissue sections by placing in 70% formamide/30% 2XSSC solution in a water bath at 81°C for 5 min. Immediately place consecutively in 70%, 85%, and 100% ethanol, Imin per vat (see Note 11). 17.45°C for no more than 2 min to dry the sections. 1. Dim the lights in the room. 2. Take IOpL of probe solution with a microsampler and add it in a linear drop on a coverslip (see Note 12). 3. Place the coverslip over the tissue and rubber cement to temporarily seal the slide. 4. Sections are placed in an airtight wet box (no water can drip off) at 37°C for 18 h (overnight). 1. Set up a water bath at 71°C and heat 50 mL of 2XSSC containing 0.3% NP-40 Cleaner. 2. Dim the lights. 3. Remove the slide from the warming chamber, peel off the rubber cement with forceps, and immerse the slide in 2XSSC/0.3% NP for several minutes at room temperature (see Note 13). 4. Place slides (no more than 4 at a time) in heated (72°C) 2XSSC/0.3%NP for 2 min. 5. Remove slides and quickly place in room temperature 2XSSC, air dry. 6. Add a drop of IOjuLDAPI to the coverslip, cover the slide with the coverslip and turn it over (see Note 14). 7. seal the sides of the coverslip with nail polish. 8. Place the slide in an airtight slide box and store at 20°C. 9. Return slides to room temperature and wipe off excess water before viewing under a microscope. 1. Browse the H&E slice under the low-power objective (10X or 4X) of a bright-field microscope to locate the area of concentration of tumor cells you wish to study (see Note 15). 2. Count 60 tumor cells from different fields of view (see Note 16). 3. Note down the red and green signals of each tumor cell (see Note 17?20). 1 We have had successful FISH with 20 year old wax blocks. 2 Tissue sectioning should be done by a skilled person using a thin-sectioning machine, always keeping the sections oriented in the same direction. 3. This step should be performed under the supervision of a pathologist. Tissue samples should be checked to determine that they contain the appropriate number of invasive tumor cells. Only invasive breast cancer cells may be counted. Invasive tumor cells must be distinguished from ductal carcinoma in situ cells, lymphocytes, histiocytes, mesenchymal fibroblasts, or adipocytes that appear on the same section. If your count includes 2/2 counted cells or expanded cells, it should be rechecked to make sure that normal tissue cells (especially exudate lymphocytes) were not counted when the tumor cells were counted. Scanning the tissue section at low magnification and labeling the tumor cell areas and markers facilitates the next step of positioning the HSH section. Markers include blood vessels, adipose tissue zones, normal ductal zones, and DCIS zones. Note the heterogeneity within the tumor when analyzing FISH sections. Note that any improper fixation or tissue distortion (especially when the sections are frozen) may interfere with interpretation. We believe that tumor regions can be drawn on coverslips of H&E sections with a Sharpie?marker for reference. 4. The optimal time for protease treatment depends on the age of the wax block and requires knowledge of the tissue composition and the quality of tissue fixation prior to HSH preparation. The duration of protease action should be reduced for specimens with coarse needle punctures, sections with fewer tumor cells, and tissues with large areas of necrosis. These samples need to be treated carefully to avoid overdigestion. 5. Meanwhile, add 50 mL of lmol/LNaSCN to the staining bath and place it in a water bath, adjusting the temperature to 81. 6. This step should be done in a fume hood. These solutions can be used on many slides and used for several months until they become dirty. 7. Simultaneously add 50 mL of 0.2 mol/L HCl to the vat and place in a 37°C water bath. Thaw one package of pepsin and mix in O.2 mol/L HC1. Prepare the following: 35 mL of formamide mixed with 15 mL of 2XSSC and 50 mL each of 70%, 85% and 100% ethanol. 8. After removing the slices, turn off the water bath until the 70% formamide and 30% 2XSSC in the vat are at 71°C. 9. Sections prepared from long-stored wax blocks (more than 2?3 years after surgery) will have a brighter signal if a second formalin fixation has not been performed. 10. Remove the probe for thawing and make sure the H&E slice is next to it for reference. 11. If using HyBritechamber (VYSIS30-144010), skip steps 16 and 17. 12. Place the coverslip on a clean brown paper towel on the bench. (eye-catching), hold the slide with the target area of the tissue section facing the probe and slowly approach until it comes into contact with the probe liquid, quickly pick up and flip the slide so that the coverslip is tightly pressed against the slide. The probe solution should diffuse rapidly under the coverslip without forming bubbles. Use a permanent marker to mark the slide with a thin line at the top and bottom corners of the coverslip. 13. The coverslip should be slipped in liquid or gently removed with forceps. The tissue is very fragile at this point and care should be taken not to scratch it. 14. For smaller areas of tumor cells we use a 5PL probe and 18 mm coverslips. vwrscientific, catalog number 48380046. 15. Find any marker and go to 25x objective. Use the fluorescence microscope to locate the target area on the H&E slice and note the position of these markers and their morphological characteristics under fluorescent light. Take this impression and look at the FISH slice again under the 25x objective and find the location of the cells. Locate the area of tumor cells and remember the characteristics of the surrounding tissue structures. Change to 100x objective and oil microscope to observe the same field of view. The tumor cells should be free of connective tissue, but still be closely packed in clusters as shown in the H&E section. 16. Using a trichrome filter (orange/green/DAPI blue), you can see that the nuclei are completely blue-stained and the probe has a bright orange spot, while the CEP17 probe has a green spot. To determine if the hybridization process is correct, analyze some mesangial cells or adjacent lymphocyte nuclei by 2/2 counting. Look for tumor cells that do not overlap and have bright, scattered signals. Look for cells with two green signals and count the corresponding red signals. 17. Overdigestion can result in discrete or roughly clumped tumor cells that lose surrounding tissue; if this occurs, reduce the protease digestion time when repeating the experiment. Poor fixation may result in "hollow cells", which are structurally intact but show dark areas in DAPI. This cannot be remedied by repeating the procedure, but by carefully reviewing the section and looking for the best preserved areas in other wax blocks for FISH. Estimate the technical quality of the slide preparation and use this to evaluate whether the sample is truly monosomic or multisomic or whether the experiment should be repeated. 18. A very weak signal can be seen through a single-channel filter, i.e., a specific nucleus is continuously observed with a tricolor filter, and the yellow and green filters are changed sequentially to identify the weaker signal under the tricolor filter. 19. If HE sections or IHC-stained sections show tissue heterogeneity, 20 cells from each region are selected for further analysis. We and other investigators (Ridofi et al.) have published articles illustrating that the experimental results for DCIS and infiltrative tumor components are different (Fig. 1E). 20. It is recommended to use the Vysis probe kit to collect red and green fluorescent signaling information from each cell. By recording individual cells it is possible to evaluate the degree of heterogeneity and the distribution of counts in the sample. When all data is given as an average, the information from individual cells is not reflected. This information becomes important when evaluating cases of low or critical amplification. For more product details, please visit Aladdin Scientific website.
Probe kit Fluorescence microscope