FISH Tips and Troubleshooting When viewing the results of a FISH assay, ensure that your microscope is properly aligned and functioning optimally. The following table lists some less than optimal results that you may encounter using the FISH probes. Probable causes and suggestions to improve assay performance are included. Problem Probable Cause Possible Solution Distorted Specimen slides dried too Increase temperature of water bath chromosome quickly during preparation. (increases humidity) used when morphology dropping slides. Decrease the temperature of the slide warmer during sample preparation. Increase drying time to at least overnight at ambient temperature, and then age slides at least 24 hours at ambient temperature. Do not bake slides at high temperature. Specimen over-denatured. Ensure the denaturation solution was made according to the package insert. Ensure temperature of denaturation solution is 73±1°C prior to immersing the slide; decrease the temperature to 72°C. Decrease the time the slide is immersed in the denaturation solution by 1–3 minutes. Specimen slides not thoroughly Warm specimen slides to 45–50°C dry prior to immersion in prior to denaturation or dehydrate denaturation solution. slides through a series of 1 minute EtOH rinses (70%, 85%, 100%). Specimen slides too fresh prior Age slides at least 24 hours at ambient to denaturation. temperature. Problem Probable Cause Possible Solution High slide Glass slides not sufficiently Immerse glass slides in EtOH and wipe background cleaned prior to sample dry using lint-free paper prior to preparation. dropping slides. Cellular debris in sample Wash cell pellet with fresh fixative preparation. three times and repeat the slide dropping procedure. Slide inadequately washed Ensure the wash solutions were made following hybridization. according to the package insert. Ensure pH and temperature of wash solutions are correct. Remove coverslip. Repeat the wash procedure. If using the rapid wash procedure, alternatively use the formamide wash procedure. Wash solutions used too Ensure wash solutions containing long or stored improperly. formamide are stored at 4°C. Discard after 7 days or frequent use. Discard all other wash solutions after 1 day. Ensure the pH of the formamide wash solutions are pH 7.0–8.0 Viewed hybridization using Switch to filters with smaller long bandpass filters. bandwidths or to multi-bandpass filters to reduce background light. Weak or no Specimen slide not Ensure temperature of denaturation signal adequately denatured. solution in the Coplin jar is 73±1°C prior to immersing the slide. Increase temperature of denaturation solution to 74°C. Increase the time the slide is immersed in the denaturation solution by 2–4 minutes. Problem Probable Cause Possible Solution Weak or no Specimen slide not adequately Contact Vysis Technical Service for signal prepared for FISH. protocols describing how to prepare a specimen for FISH. Specimen slides improperly Age for 24 hours at ambient aged after dropping specimen. temperature before performing FISH on them or put in 2XSSC for 2 min. at 73C. Specimen slides not thoroughly Warm specimen slides to 45–50°C dry prior to immersion in prior to denaturation or dehydrate denaturation solution. slides through a series of 1 minute EtOH rinses (70%, 85%, 100%). Probe not added. Prepare a new probe mixture. Allow the probe to thaw completely. Vortex or pipet reagents to mix; centrifuge briefly. Pipet probe slowly. Probe, hybridization buffer, or Vortex or pipet reagents to mix; probe mixture were not mixed centrifuge briefly. well prior to use. Probes improperly diluted for Use the volumes stated in the assay hybridization. procedure to maintain the ratio of the probe mix (7 µL hybridization buffer: 1 µL probe: 2 µL purified H2O). Ensure the pipet is calibrated. Allow hybridization buffer to thaw completely and to reach ambient temperature prior to use; pipet slowly. Probe not adequately denatured. Ensure temperature of the water bath NOTE: Does not apply to used to denature the probe mix is probes that are supplied in 73±1°C. hybridization buffer and Denature the probe mixture for 5 denatured. minutes. Problem Probable Cause Possible Solution Weak or no Probe not applied to the target Plan so the probe is applied signal sample immediately after the immediately after the slides are probe was denatured. removed from the 100% EtOH solution. Ensure the EtOH has evaporated before applying probe. Remove tube containing probe mix from 73±1°C water bath and immediately place the tube on a 45– 50°C slide warmer. Keep the tube on the slide warmer while pipetting the probe onto the slide. Process only as many slides as you can and still maintain the correct temperatures and times according to the assay procedure. Air bubbles were trapped under Apply coverslip by first touching the the coverslip during surface of the probe mixture. hybridization. Place the slide with the coverslip down on a blotter and very gently press out visible bubbles. Wash conditions or solutions Ensure that the wash solutions were incorrect. made according to the package insert. Ensure the temperatures of the wash solutions are at the stated temperatures for the wash procedure followed. If using the formamide wash procedure, ensure the pH of the wash solutions are pH 7.0–8.0. Ensure that the thermometers and pH meters used are calibrated properly. Remove coverslips before immersing slides in wash solution. Problem Probable Cause Possible Solution Weak or no Probes or specimen slides Store undiluted probe at –20°C signal stored improperly. protected from light. Store non-hybridized slides desiccated at –20°C for an extended period or at ambient temperature for short periods. Store hybridized slides at –20°C protected from light for up to 6 months. Wrong counterstain used. Remove coverslip. Immerse slides for Counterstain is too bright. 5 minutes in 2X SSC/0.1% NP-40 at ambient temperature; dehydrate slide through a series of 1 minute EtOH rinses (70%, 85%, 100%). Air dry and reapply counterstain. Viewed hybridization using Multi-bandpass filter sets provide less inappropriate filter set. light than single bandpass filter sets, so probe signals may appear fainter when viewed through the multi- bandpass sets. Use correct filter for viewing the probe fluorophore. Contact Vysis Technical Service for further information. Microscope configuration or Contact your microscope objectives not adequate for manufacturer. viewing FISH results, or microscope filters are damaged. Low signal Probes diluted inappropriately; Ensure the probe mixture was made specificity often too much probe in the according to the package insert. assay. Wash temperature too low. Maintain the wash temperature of the wash solutions by placing no more than four slides in one Coplin jar at a time and ensuring that the temperature of the wash solution is correct before washing another set of slides. Problem Probable Cause Possible Solution Low signal Wash solution stringency too Ensure the wash solutions were made specificity low. according to the package insert. NOTE: The lower the concentration of salt (SSC), the higher the concentration of formamide and NP- 40, the more stringent the wash. Bright or weak Counterstain appears weak: Remove coverslip. Immerse slides for counterstain specimen slides not dehydrated 5 minutes in 2X SSC/0.1% NP-40 prior to applying counterstain or at ambient temperature; dehydrate oil droplets in counterstain. slide through a series of 1 minute Wrong concentration of EtOH rinses (70%, 85%, 100%). Air counterstain. dry and reapply counterstain. NOTE: DAPI I counterstain is If counterstain appears too bright, eight times more concentrated dilute the counterstain in antifade than DAPI II counterstain. solution (Order No. 32-804029) before applying. Counterstain too old or exposed Store counterstain at –20°C protected to light for extended periods. from light and when using. Ensure the counterstain is not used past the expiration date.