Oxford Gene Technology The Molecular Genetics Company

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1 Oxford Gene Technology The Molecular Genetics Company UK UGM Pathology Workshop Troubleshooting FISH Alex Hobbs Field Application Specialist-Cytocell 1

2 Overview PETS FISH common issues Analysis considerations 2

3 Protocol Overview FFPE Preparation Sample Quality De-paraffinisation Pre-treatment Enzyme digestion Probe application Denaturation Hybridisation Hybridiser or incubator Post hybridisation washes ph 7+/-1 Temperature 0.4xSSC 72 C Counterstain and analysis 3

4 Most common issues encountered 1. Pretreatment and enzyme digestion conditions. 2. Poor performing water bath. 3. Inconsistent denaturation using a hybridiser. 4. Poor and degraded microscope filters 5. FFPE handling pre-fish. 4

5 Sample quality considerations Age of FFPE block is critical. Blocks older than 2 years with produce poorer FISH results. Positive cells are generally localised in a very small region (typically 28% of Tissue mass). Inadequate fixation, formalin is preferable. Processing artefacts, fine black precipitate. Blunt microtome blade leads to shearing. Eosin flare effect. The tissue will fluoresce green across the tissue, if the block has been dotted with Eosin (histology counterstain). Decalcification of tissues with 10% formic acid. Good communication with Histology is critical! 5

6 Factors affecting fixation ph. Ideally between 6-8. Formalin with phosphate buffer. Penetration of fixative. Again Formalin is the best or thinner sections (2-3uM). Fixative volume. A 10:1 ratio of fixative to tissue ideal. Agitation can assist. Temperature. Pre-heated formalin best. Concentration. 10% Formalin. High concentration Pro-longed tissue drying prior to fixation. 6

7 Problem 1 Microtome sectioning Cause Sections tear very easily and do not slice well. Solutions Place tissue block on ice for 20mins before sectioning. This will ensure smooth cutting of the material. Use a sharp cutting blade. These should be replaced regularly. Longer, continuous ribbons are easier to manipulate. 7

8 Problem 2 Slide Mounting Technically difficult, requires practice and experience. Solutions Ensure the water temperature does not exceed the melt temperature of the paraffin wax. 40 C should be sufficient. Use scalpels and/or tweezers to manipulate the sections. Super frost plus, positively charged slides will ensure good adhesion. 8

9 De-paraffinisation I Cytocell use Histoclear (x3 washes at RT for 10mins each). Clearing agent Histoclear/ Haem-de Pros -Excellent at preserving tissue -Safer -Affordable Cons -Requires more washes Ultra Clear Xylene -Odourless, less toxic and flammable -Cost effective -Rapid tissue penetration -Requires more washes -Tissue distortion -Highly toxic 9

10 Problem 3 Excess Paraffin Cause Excess paraffin will preferentially bind to FITC. Insufficient paraffin clearing effects the probe s ability to penetrate the tissue Solution Ensure all the paraffin is cleared. More washes for longer times 10

11 Pre-treatment Ensure that both the solution and enzyme are placed in a fridge 2-8 ºC on arrival. Its role is to break the formalin induced disulphide bonds This is most critical step in the protocol The water bath temperature should ºC and be maintained for min 15mins. Customer hints and tips The solution will expand in the jar when heated, be careful not to fill to the very top. Replace the pre-treatment solution after every batch (max 20 slides per wash). LPS0100 Pre-treatment kit 11

12 Pre-treatment 12

13 Enzyme digestion This is a very critical step in the protocol Use a start digestion time of 10mins. The user needs to optimise their own times depending on the tissue section used. Remove the enzyme solution from the fridge 10-15mins before use allowing it to reach RT. Carry out this step ideally on a 37 ºC hotplate, this will ensure optimum enzyme activity. User hints and tips Do not leave the solution out for longer, this can reduce the enzyme activity over time. This can ultimately lead to weak probe signals. Apply a liberal amount of enzyme solution over your target area. A small but sometimes significant level of evaporation will occur. The enzyme solution will appear to receed from the edges. This can effect FISH consistency across the slide. 13

14 Enzyme digestion 14

15 Problem 4 Over digestion Features -Autofluorescence -doughnut or ghost nuclei -absence of DAPI staining in some areas -destroyed tissue morphology -loss of target gene signal in some cells Solution -Decrease enzyme digestion time. 15

16 Problem 5 Under-digestion Features -A cloudy haze across the cells. -Inconsistent DAPI staining. -Reduced probe signal strength. Solution -Increase enzyme digestion time. -Check the enzyme digestion. DAPI stain and look under microscope before proceeding. 16

17 Optimised pre-treatment and digestion times Tissue type Pre-treatment time (mins) Enzyme digestion (mins) Breast Lung Ovary Kidney Colon Schwann cells (nerve tissue) Brain

18 Probe and target co-denaturation Use 8-12uL of probe solution. Increase this volume for larger sections Choose an appropriate size coverslip (tissue size dependent). This ensures good spreading of probe across the target area. Ensure a good seal with rubber glue (PCA005), preventing evaporation. User hints and tips Check the hotplate temperature, using the Cytocell slide thermometer (PCN002). It s a quick, visual check to verify the hotplate temperature The denaturation temperature is 75 C however increasing to 80 C can improve signal strength (consult tech support). 18

19 Hotplate vs automatic hybridiser Pros -Immediate co-denaturation -Better regulation of temperature Cons -For high-throughput slide processing not ideal Pros -Less manual input, easy to use. -High throughput Cons -Temperature inconsistencies across the positions. -Loss of humidity -Ramp time to temperature 19

20 Problem 6 Over-denaturation The effects of over-denaturation are tissue fragmentation and increased background Ovary (85 C for 7mins) Solution Co-denature at 75 C 5mins 20

21 Problem 7 Under-denaturation This example Skin biopsy Co-denaturation in a 75 C hybridisation oven. Weak probe signals Solution Co-denaturation on a hotplate/thermobrite at 75 C 21

22 Hybridisation Use a sealed, darkened, moist (40-45% humidity) hybridisation box Use a hybridisation oven that can maintain 37ºC Check the temperature using a thermometer daily. User hints and tips If using a Thermobrite/Hybrite. Ensure that all the slide positions are calibrated for temp and humidity The strips are pre-moistened in a 37 C water bath before use. Can produce control probe (FITC) drop out. 22

23 Post hybridisation washes PCA002 20xSSC Cytocell supplied Ensure that the solutions are made fresh (max 7 day expiry) Tween 20 is milder and NP40 (Igepal) is good to use User hints and tips Ensure that the ph is 7 ±1 Ensure that the temperature of the 0.4xSSC solution is 72±1 degrees The results can mean higher background (less stringent) or loss of probe signals 23

24 Problem 8 Too high stringency Control Stringency too high Solution -Re-make 0.4xSSC ensuring that the ph=7 and the wash temperature is 72+/-C 24

25 Problem 9 Low Stringency Control Stringency too low Too low stringency will produce high background as non-specific bond probe is not removed. Solution -Re-make 0.4xSSC ensuring that the ph=7 and the wash temperature is 72+/-C 25

26 DAPI Counterstaining Cytocell supplied ES DAPI works very well. Use 8-12uL per target area, increase volume if necessary. Following counterstaining, allow min 10mins before visualising User hints and tips After counterstaining place slides in a -20 C freezer for 20-60mins. This can make the signals appear sharper, more distinct. This has been tested internally and makes a difference Liquid DAPI protocol using a Vectashield wash before applying the DAPI. This is very good for looking at the tissue morphology, however can compromise the signal. 26

27 Problem 10 Higher concentration DAPI This is a trade off, for tissue FISH, the cell morphology will be very good but will produce diminished probe signals Solution -For Pathology FISH, this is at the discretion of the user. If the protocol is fully optimised and producing good signals, high concentration DAPI is acceptable. 27

28 Microscope We recommend using a dual or triple filter for PETS FISH analysis Single Texas Red, FITC and aqua are useful for signal confirmation. User hints and tips Ensure that the filters are checked for degradation regularly (monthly). Signals can appear weaker with higher background Check the age of the bulb (we recommend replacing at 200 hours). Avoid repeatedly turning on and off the bulb. This decreases the bulb life. 28

29 Analysis considerations LPS016 MDM2 amplification Sarcoma LPS014 SYT breakapart Sarcoma -High background. This is endemic in all Tissue sections, the overall thickness of the tissue matters. 29

30 Analysis considerations I -Signal and Nuclear truncation. Optimising for the correct thickness 2-4uM will make a difference. -Truncation effect is more noticeable in thinner sections and can generate a high false positive rate. <30% loss of a signal can be attributed to signal truncation -Mixed cell populations, carefully screen the whole slide -Avoid scoring large macrophage cells -Probe signal distance -Check that the appropriate filter set is used. -To confirm signals enumerate on a single filter, consider focal planes and split signals. Avoid scoring cells on the periphery. -General scan of the area to assess the hybridisation efficiency (~85%), to identify good areas. -Count/score signals on a single filter, deletions should be marked, tissue heterogeneity noted. -When selecting cells they need to be of uniform shape, good DAPI staining and cells separated. 30

31 Summary points Tissue FISH has multiple steps where errors can be introduced. LPS0100 IFU should be followed in conjunction with Cytocell probes. Xylene or Histoclear work well and should be recommended. Correct and calibrated equipment is essential! Especially water baths and hybridisers. Each lab needs to optimise their own pre-treatment and enzyme digestion times, depending on tissue type. Technical support available from Cytocell. 31

32 Questions? 32