Chromogenic in situ hybridization and FISH in pathology
Chromogenic in situ hybridization and FISH in pathology
Fluorescence in situ hybridization (FISH) techniques enable rapid detection of chromosomal abnormalities in a variety of tissues, including fresh and archival specimens. These techniques have been widely accepted by clinical cytogenetics and research organizations. However, these methods are not well used in pathology services for non-cytogenetic diagnosis, in part because FISH imaging equipment is not universally available to diagnosticians (surgical pathologists) responsible for histologic diagnosis. Therefore, the improvement of various in situ hybridization probes and assays has been satisfactory, especially in the last 5 years, and it has become possible to perform routine analyses by enzymatic in situ hybridization through a light microscope.
Operation method
Chromogenic in situ hybridization and FISH in pathology
Materials and Instruments
Tris-EDTA solution PBS Digest-All 3 formalin phosphate buffer Xylene Ethanol Biotin- and digoxin-labeled DNA probes CAS-block (Zymed Laboratories) Streptavidin Move I. Slide treatment before hybridization 1. Bake the slides in a 60°C oven overnight. 2. Deparaffinize in xylene 3 times for 15 min each time. 3.100% ethanol dehydration 3 times, 2 min each time, air drying. 4. Heat a plastic dye vat containing Tris-EDTA or other plastic container for microwave water to 199°F in a microwave oven. 5. Place the slide in the Tris-EDTA and continue heating in the microwave at 199°F for 15 minutes. 6. Remove the slide and place it in PBS. 7. Blot excess PBS from slide and place in a 37°C humid slide box (or other humid incubator). 8. Add 100-500uL Digest-All3 to the slide and digest for 10-30 min depending on tissue type and fixation (see Note 1). 9. Place the slide in PBS to terminate digestion. 10. Fix the slides in 10% formalin buffer for lmin, and then remove the formalin by gently washing the slides in a staining vat containing 40 mLPBS. 11. The slides were dehydrated in 70%, 85% and 100% ethanol for 2 min each at room temperature and dried in air. 1. Add enough probe, e.g., 10uL, to the tissue section to fit the size of a 22 mmX22 mm coverslip range. 2. Place the coverslip onto the section and seal the edges with rubber cement. 3. Place the slide in the slide slot of the thermal cycler and denature the slide at 94°C for 3 min. 4. Transfer the slides to a slide box and place them in a humidified incubator at 37°C overnight. 1. Set the temperature of the water bath according to the number of slides in each vat, 73°C for 1 slide, 74°C for 2 slides, 75°C for 3 slides, and 76°C for 4 slides. 2. Place a staining vat containing 40 mL of 0.5XSSC in the water bath and equilibrate the temperature of the vat with the temperature of the water bath. The vat and contents should be placed in the water bath when the temperature of the vat and contents are not below room temperature. Otherwise the vat may blow up. Use a thermometer to determine the final temperature. 3. Remove the emery and coverslip from the slide. 4. Elute the slide in 0.5XSSC for 5 min. 5. Transfer the slides to a PBS/T staining vat containing 40 mL of PBS/T at room temperature. 1. Remove excess PBS/T from the slide and place it in a moist plastic slide box. 2. Add 100~500uL CAS-bl ○ Ck antibody dilution to the slides and incubate for lOmin at room temperature. 3. Gently dip off the CAS-block, then add 100~500uL of fluorescence-conjugated detection reagents, such as 1:500 dilution of streptavidin-Alexa594 and anti-digoxigenin-incendrin. Incubate for 30 min at room temperature. 4. Elute the slides in 40 mLPBS/T for 3 times at room temperature for 2 min each time. 5. The slides were re-stained with DAPI sealing medium. 1. Remove excess PBS/T from the slide and place it in a moist plastic slide box. 2. Add CAS-block to the slides and incubate for lOmin at room temperature. 3. Gently remove the CAS-block, add HRP-Streptavidin and incubate for 30 min at room temperature. 4. Elute the slide in 40 mLPBS/T for 3 times at room temperature, 2 min each time. 5. Prepare DAB substrate according to the instruction guide, remove excess PBS/T from the slide and incubate the slide with DAB for 15 min. 6. Elute the slides in 40 mLPBS/T for 2 min three times at room temperature. 7. Remove excess PBS/T from the slides, add anti-decorbutin-fluorescein (see Note 2) and incubate for 30 min at room temperature. 8. Elute the slides in 40 mLPBS/T for 2 min three times at room temperature. 9. Remove excess PBS/T from the slide, add alkaline phosphatase anti-luciferin and incubate for 30 min at room temperature. 10. Elute the slides in 40 mLPBS/T 3 times for 2 min each at room temperature. 11. Prepare Fast Red substrate according to the instruction manual and filter through a 0.45pm membrane. 12. Stream/gently dip the slide in PBS/T, then add the Fast Red Substrate to the slide and incubate for 30 min, changing the Fast Red Substrate twice at IOmin intervals, i.e., gently dipping the slide in the Substrate and adding the newly filtered Substrate. 13. Elute the slides in 40 mLPBS/T at room temperature. 14. Restain slides with hematoxylin, being careful not to overstain (see Note 3). Gill'sformula (non-organic solvent-see Note 4) should be used in combination with Fast Red. 15. Rinse the slide under running water for a few minutes and do not enhance the blue color of hematoxylin with ammonia citrate. 16. Warm the glycerol gel in a 50-75°C oven or water bath, place a drop on the section (without organic solvents) and cover with a coverslip. Gently press the coverslip to remove excess glycerol gel. If the glycerol gel has solidified, the slide can be heated on a hot plate (37-45°C). 1. Residual extracellular and intracellular proteins can cause autofluorescence between a pale red-orange color, which diminishes the FISH signal, especially when the true probe signal is weak. Autofluorescence can be reduced by increasing the enzymatic digestion time of paraffin sections. Inadequate digestion of tissue sections will also prevent the probe from entering the target chromosomal region (making the 'true' signal weaker) and non-specific binding of the probe to non-chromosomal cellular components. Therefore, in general, the digestion time should be increased if the cell morphology is still very good (based on DAPI restaining) and the cells have scattered small non-specific signals. Properly hybridized paraffin sections should have well-preserved cell morphology (although not necessarily very good), very distinct probe signals, and no nonspecific signals. 2. The CISH assay procedure herein is achieved by subsequent incubation with fluorescein anti-digoxin and alkaline phosphatase anti-luciferin for amplification of digoxin-labeled probes. However, digoxin-labeled probes can also be detected without an amplification step, in which case alkaline phosphatase anti-digoxin is used instead of fluorescein anti-digoxin. 3. Compared to the fluorescein and rhodamine fluorescence of two-color FISH, the difference between the different colors of two-color CISH is not obvious, and it is particularly difficult to distinguish DAB from fast red when the probe signal is very small. However, it is often possible to discriminate the staining by adjusting the focus of the section up or down. At the exact point of focus, Fast Red is more or less refractive and the red color is obvious. It is also important that the hematoxylin restaining is done in the smallest amount that allows the nucleus to be seen. Thus, hematoxylin should be only a light blue color. Otherwise, hematoxylin will obscure the DAB that is distinguished from the fast red color. 4. Coverslips should not be sealed with organic solvents after Fast Red staining. Although we have found that CISH slides can be stored at room temperature for approximately several months, FISH and CISH slides are routinely stored at 4°C. The FISH signal will be partially quenched during storage, whereas the CISH signal is stable for approximately 2 years. 5. It is possible to combine CISH and immunohistochemical staining with a slight modification of the two-color CISH procedure (Fig. 1). In this case, a chromosomal target and a protein can be detected by single-color CISH and immunohistochemistry, respectively. For more product details, please visit Aladdin Scientific website.
Paraffin sections Oven Microwave oven Staining cylinders Thermal cycler Humidity oven