Specific DNA fluorescently tagged PROTOCOLO DE UTILIZACIÓN

Transcription

1 Specific DNA fluorescently tagged PROTOCOLO DE UTILIZACIÓN Product: 1. DNA probe fluorescently labeled. 2. Hybridization Buffer 3. Reagents to prepare Mounting Solution (DAPI + antifade) These probes are designed to be utilized on interphase or metaphase cells obtained through standard citogenetic procedures, see: The ACT Cytogenetics Laboratory Manual. 2nd ed. New Cork: Raven Press; Materials required but not provided Ultrapure Formamide solution* 2XSSC* Ethanol solutions 70, 90 y 100% Pepsin* Non-ionic detergent Counterstain solution* diluted in antifade* ph-meter or ph indicator papers Coverslips Wash solutions (1 y 2)* Rubber cement 0,2 or 0,5ml PCR tubes Coplin jars Moist chamber Calibrated thermometer *see prepatation instructions below. Equipment Required: Fluorescence microscope: Not always a microscope used in immunohistochemistry reactions or other fluorescent analysis is appropriate. The requirements to properly observe FISH reactions are as follows o Excitation Source: a 100 Watts mercury lamp with a lifespan of 200 hours is recommended. It is necessary that once positioned, the lamp is correctly aligned. o Objectives: To target location (interphases or metaphases) objectives of 10, 20 and / or 40X are recommended. The reaction analysis requires a special fluorescence immersion objective with a numerical aperture 0.75 o Excitation-emission filters: Each fluorochrome require different filters. The requirements for our probes are listed in the table below: Fluorochrome Excitation(nm) Emission (nm) Green Red DAPI

2 Microcentrifuge Microliter pipettor for 1 to 10 µl volumes Vórtex Timer Water bath Incubator Reagent preparation: 2XSSC: Sodium chloride (NaCl) 300mM Sodium citrate dihydrate (Na 3 C 6 H 5 O 7 ) 30mM Adjust ph to 7.0 with drops of Hydrochloric acid (HCl) 1N Denaturation Solution: Ultrapure Formamide 70% 2XSSC 30% Adjust ph:7-8. Store at 4 C. Discard after 7 days. Pepsin solution: Dilute 0,5mg of pepsin in 1ml Hydrochloric acid (HCl) 0,01N. Wash solution 1: In a coplin suitable for 80ml, add 16ml of 2XSSC, 64ml distilled water and ml of non-ionic detergent. Wash solution 2: In a coplin suitable for 80ml, add 80ml of 2XSSC and 0.80 ml of nonionic detergent. 2

3 Cautions and warnings (Read carefully): Analytical reagent. For research use only. This product is NOT tested for diagnostic or therapeutic use. The reactions and the interpretation of them must be performed by qualified personnel properly trained. All biological samples should be treated as potential carriers of infectious agents. Avoid exposure of sample to acid or alkali in high concentration as well as extreme heat. These factors can damage DNA and cause failure of the FISH reaction. The failure or omission of any step of the protocol detailed below can lead to erroneous or unacceptable results. The hybridization buffer containing formamide, a teratogen and therefore should avoid contact with skin and mucous membranes. It is recommended the use of gloves and jacket work throughout the process. All hazardous materials should be discarded according to rules of your institution. 3

4 Utilization Protocol Preliminary considerations: During the aplication of this protocol It is no nesessary to work in reduced-light conditions. LIVE probes were developed to not lose their fluorescence during short periods (hours) of exposure to artificial light. While it is highly recommended to keep at -20 ºC, the probes Live does not lose its activity after 48hrs at room temperature. The Hybridization Buffer provided leads to results in 30 minutes of hybridization. A hybridization time over 30 minutes does not affect the quality of the reaction. It is recommended to use 45 C for 30 minutes to about 8 hours hybridizations. If the reaction will be revealed next day the temperature should be 37ºC Cellular Smear For the cellular smear it is very important not to use extreme heat(flame, flame-drying, etc.). Pre-treatment In general Live probes works well without any pre-treatment, however the presence of cellular debris or hybridization on certain samples as buccal mucosa, amniocytes, etc. require to clean the target DNA to improve the hybridization. Over the smear sample apply a drop of Pepsin solution. Add a coverslip. Incubate in moist chamber at 37 C (times vary with the severity of the treatment, with a minimum 5 minutes). Discard the coverslip and rinse briefly under running water. Dehydrate in ethanol series: 70, 90 and 100% 1 minute each. Allow to dry completely 4

5 Fluorescent in situ Hybridization reaction Protocol 1: Denaturation using Formamide Probe preparation: Thaw the probe tube, mix on Vortex Thaw the hybridization buffer In a PCR tube add: 7µl of hybridization buffer 1µl of probe Mix in vortex Denaturation: NOTE: At least 30 minutes before denaturing, heat the denaturation solution to desired temperature. With a calibrated thermometer corroborate the temperature inside the coplin. On the back of the slide highlight the region to hybridize (the 8µl prepared cover approximately a region of 22x22mm). Immerse the slide in the denaturation solution at C for 1-5 minutes (depending on the age of the smear) Using tweezers remove the slide and immediately immerse in 70% ethanol agitating for a few seconds to eliminate remaining formamide. Dehydrate in ethanol 70, 90 and 100% 2 minutes each. Allow to dry completely. Denature the probe solution at a minimum temperature of 70ºC 5 minutes. This can be done in a temperature-controlled device or simply leaving the tube in boiling water in a polystyrene support. Place the probe solution on ice a few seconds. Centrifuge briefly to collect the evaporated fraction. Add the probe solution on the region to hybridize Place a coverslip of appropriate size. Seal the edges with removable contact cement or place a sheet of Parafilm that exceeds the size of the coverslip. Hybridization: LIVe hybridization buffer gives excellent results with any probe in just 30 minutes of incubation at 45ºC Place the mounted slide in moist chamber at 45ºC. Incubate for at least 30 minutes at 45ºC or overnight at 37ºC. 5

6 Protocol 2: Denaturation with Sodium Hydroxide (NaOH) This protocol optimally preserves chromosome morphology, allows FISH reaction the same day of the cellular smear preparation and provides high quality results. Furthermore, according to the quality of the cell suspension is possible to observe a banding pattern similar to G banding. NaOH can sometimes generate autofluorescence on nuclei and / or chromosomes. In these cases it is recommended to slightly age the smears at 37-45ºC 2 hours. Probe preparation: Thaw the probe tube, mix on Vortex Thaw the hybridization buffer In a PCR tube add: 7µl of hybridization buffer 1µl of probe Mix in vortex Denaturation: On the back of the slide highlight the region to hybridize (the 8µl prepared cover approximately a region of 22x22mm). Immerse the slide in a solution of NaOH 0.1M in Ethanol 70% at room temperature for 6 minutes. Using tweezers remove the slide and immediately immerse in 70% ethanol agitating for a few seconds to eliminate remaining NaOH. Dehydrate in ethanol 70, 90 and 100% 2 minutes each. Allow to dry completely. Denature the probe solution at a minimum temperature of 70ºC 5 minutes. This can be done in a temperature-controlled device or simply leaving the tube in boiling water in a polystyrene support. Place the probe solution on ice a few seconds. Centrifuge briefly to collect the evaporated fraction. Add the probe solution on the region to hybridize Place a coverslip of appropriate size. Seal the edges with removable contact cement or place a sheet of Parafilm that exceeds the size of the coverslip. Note: Once the above process, a good alternative is to place the slide on a hot surface at 71 C for 4 minutes. Usually this extra step gives better results, although it may slightly decrease the quality of chromosome morphology Hybridization: LIVe hybridization buffer gives excellent results with any probe in just 30 minutes of incubation at 45ºC Place the mounted slide in moist chamber at 45ºC. Incubate for at least 30 minutes at 45ºC or overnight at 37ºC. 6

7 Protocol 3: Co-denaturation This protocol has been designed to unify co-denaturation time and temperature. Here, these parameters are the same for cell smears prepared on the day or kept at -20 C. Probe preparation: Thaw the probe tube, mix on Vortex Thaw the hybridization buffer In a PCR tube add: 7µl of hybridization buffer 1µl of probe Mix in vortex Co-Denaturation: On the back of the slide highlight the region to hybridize (the 8µl prepared cover approximately a region of 22x22mm). Add the probe solution on the region to hybridize Place a coverslip of appropriate size. Seal the edges with removable contact cement or place a sheet of Parafilm that exceeds the size of the coverslip. Place the mounted slide on a hot surface (hybridizer, thermostatic plate, etc.). Sometimes it is advisable to place a drop of water on the hot plate to optimize the temperature transfer. o A treatment of 15 minutes at 71 C has functioned optimally and independently of the age of the smears. Hybridization: LIVe hybridization buffer gives excellent results with any probe in just 30 minutes of incubation at 45ºC Place the mounted slide in moist chamber at 45ºC. Incubate for at least 30 minutes at 45ºC or overnight at 37ºC. 7

8 Washing: Note: At least 30 minutes before washing, heat the wash solution 1 to 71 C (+/-1ºC). With a calibrated thermometer corroborate the temperature inside the coplin. Thaw wash solution 2 to room temperature. Remove the slide from moist chamber. Carefully remove the rubber cement or Parafilm sheet. Immerse the slide in a 2XSSC solution at room temperature until the coverslip falls off. Immerse the slide in the wash solution 1 for exactly 2 minutes. Immerse the slide in the wash solution 2 for at least 1 minute. Take off the slide and drain excess liquid slightly. Place on a drop of mounting solution on the hybridized region. Place a coverslip of appropriate size Drain excess mounting solution by pressing gently and evenly with paper towels. Remove any air bubbles by pressing gently with a tip. The reaction is ready for analysis 8

9 Protocol for use (Protocol #1) Cellular smear Probe preparation DNA Probe + Hybridization buffer Denaturation Formamide 70% Dehydration in ethanol Denaturation 70ºC or more, 5 minutes Hybridization 45ºC (30 minutes to about 8 hours) 37ºC (Overnight) Washes Mounting DAPI + Antifade Analysis 9

10 Protocol for use (Protocol #2) Cellular smear Made the same day (aged 2 hours at 37ºC) Probe preparation DNA Probe + Hybridization buffer Denaturation Sodium Hydroxide 0.1M 6minutes Room Tº. Dehydration in ethanol Denaturation 70ºC or more, 5 minutes Alternative 71ºC, 4 minutes Hybridization 45ºC (30 minutes to about 8 hours) 37ºC (Overnight) Washes Mounting DAPI + Antifade Analysis 10

11 Protocol for use (Protocol #3) Cellular Smear Made the same day or Kept at -20ºC Probe preparation DNA Probe + Hibridization buffer Co-denaturation 71ºC 15 minutes Hybridization 45ºC (30 minutes to about 8 hours) 37ºC (Overnight) Washes Mounting DAPI + Antifade Analysis 11