• Make sure you do all the labwork in a laboratory equipped for work with radioactivity. Locate all containers for disposal of radioactive waste.
• Always use appropriate safety precautions, such as: gloves, labcoat and radiation screens.
• Wear your dosage meter.
• All pipette tips, tubes and other disposables should be autoclaved or certified RNA:ase free.
• Isotope, 33P, bound to ATP [Deoxyadenosine 5'-alpha-??? Triphosphate] is obtained from e.g. NEN and stored at -20°C (or lower) in lead boxes as 6µl aliquots.
• Our oligo probes are kept outside the isotope-lab in the oligo fridge/freezer. (undiluted solutions from the manufacturer and stock solutions (400ng/µl) are stored at -20°C, while working solutions (40ng/µl) are kept at 4°C).

• Hybridisation solutions are kept frozen in the isotope lab.
• The isotope lab fridge contains only isotope or isotope labelled probe

• Heat waterbath to 37°C
• Put tube with 6µl isotope aliquot on ice

• 1µl  probe (40ng/µl): labelling the tubes is a good idea at this point!
• 1.25µl ddH2O (needed?)
• 2.5µl 5xCobolt buffer  (1.25 µl 10xCobolt buffer) Should not be kept thawed for extended periods
• 3 µl TdT [3'-terminal deoxynucleoside transferase]. Do not thaw!

• Spin down, mix only with pipette
• Incubate at 37°C in waterbath for 2-3 h.

• Add STE buffer to labelled probe to make a total volume of 50µl
• Vortex the G50-column
• Unscrew cap a quarter turn and snap off the bottom closure
• Put the column in a 1.5ml eppendorf tube and pre-spin for 1 min at 735g (2800 rpm IEC 8.35cm microcentrifuge)
• Place column in a new 1.5ml eppendorf tube and apply the 50µl of sample (take care not to damage the column)
• Spin for 2 min at 735g (the purified sample is collected in the tube)
• Add STE-buffer to final volume of 200µl

• Take out the slides (from freezer) and let them dry (4-6h)
• Heat waterbath to 42°C (37°C)
• Make sure incubation "oven" is switched on at 42°C.

• 90 µl cocktail
• 5 µl 10µg/µl salmon
• 4 µl 5M DTT

• Heat hybridisation "cocktail" for 60min in waterbath; leave the foil wrapping on the tubes
• Boil salmon for 5 min, and then keep it on ice
• Thaw 5M DTT
• Prepare the cocktail, salmon and DTT-mix in a 15ml falcon tube and keep it at 42°C

• Add 2 µl probe/101µl total solution to separate tubes (1.5ml)
• Finish the hybridisation solutions by adding appropriate amounts of cocktail, salmon and DTT-mix to probe tubes.Return the finished solutions to 42°C

• Carefully vortex the hybridisation solutions. No bubbles!
• Keep solutions at 42°C until they are applied to the slides

• Prepare incubation boxes with tissue strips soaked in ddH2O. Don't overdo it -condensation will form and can dilute your solutions!
• Apply hybridisation solutions, 150µl/slide. No bubbles!
• Cover with parafilm pieces, avoid catching bubbles or wrinkling the film

• Seal incubation box with tape
• Incubate overnight (12-18 h) at 42°C

• Heat waterbath to 55'C
• Heat bottles of 1X SSC in waterbath. Estimate usage to 2 liter 1X SSC/rack of 25 slides
• Remove the incubation box with the slides from the incubator oven
• Separate parafilm from slides in heated 1XSSC in a suitable beaker. If done carefully the film should separate promptly. Note that pulling the film off is not needed and should be avoided. Remember to dispose of this contaminated solution appropriately
• Place slides in racks in 1X SSC at 55°C (contained in the beakers designed to hold the racks)
• Rinse slides 4x15 min in 1X SSC at 55°C
• Cool the rinsed slides in 1X SSC, at room temperature; cover with Al-foil (30min)
• Dehydrate:

• Dry slides covered with Al-foil; can be kept up to 24 hours at room temperature

1. Mount slides in exposure casettes. Add labelling markers to allow for evaluation of labelling.
2. Remember to add support slides to ensure an even support for the film.
3. Write a key for the placing of the slides in the cassette (add the date and planned date for developing the film)

1. Place KODAK BioMax MR film gently on slides; rough surface down, glossy up. Notch at upper left. Film is kept in light secure container in fridge. Open container only in dark room.
2. Close cassette. Locks should snap into place.
3. Remove closed cassettes from dark room and leave to expose at room temperature.

• Take cassette to the dark room.
• Use only Na-illumination
• Make sure sufficient developing and fixation fluid is available. Check fluid levels: surface levels should allow film to be sufficiently submerged.
• Start rinse water flowing -also cools fix and developer.
• Open casette and remove film; place it in metal frame.

1. Place frame in developer (20°C): 4 minutes (5 min)
2. Transfer to rinse (room temp: 24°C): 30 seconds
3. Place in fixative  (24°C): 7 min (5-10 min)
4. Transfer to rinse (room temp: 24°C): 10 min (5-10 min)

Dipping emulsion: NTB2, kept at 4°C in Hökfelt lab- undiluted emulsion in black plastic can in box/leadcase; diluted in lead cylinders; Blue gel- heated cabinet in Hökfelt lab. Waterbath for heating emulsion is stored in dark room. Other stuff...

Preheat dipping emulsion 40-45°C for 60-90 min in the waterbath. Emulsion is in gel-form at 4°C but liquid at 40°C.
Dilute dipping emulsion if not done: make sufficient volume for experiment at dilution 1+1 with ddH₂O.
Ta tillbaka dipemulsion till kylskåp i ISH/EM-lab.
Ta med all utrustning som behövs för dip (dipvanna, mätkolv, glasbägare, kleenex, handskar, underläggspapper, slides- noga sorterade efter diptid!!!) och gå in i mörkrum.

Fill small plastic dipping cup and keep it heated in the waterbath. Emulsion volume should be sufficient to dip slides...

• Dip 3 empty ISH-coated glass slides.
• Let dry 1h
• Develop

b.)  Dippa- fyll dipvanna med dipemulsion (låt stå i vattenbad under hela dipsessionen!!) ca. 5mm från övre kant; dippa 2 glas åt gången liggande rygg mot rygg, i 10s ; för glasen ned samt upp i jämnt lugnt tempo; dutta glas försiktigt mot kleenex för att få bort excess av dipemusion på distala kanten; för försiktigt isär glasen och torka av dem på baksidan; lägg sedan glasen horisontellt på isfyllda stålboxen (à torkar snabbare); överför sedan glasen till pappflak för definitiv torkning (tar 2-3 timmar); var noga med att fylla på dipemulsion efter hand.Då man går ut ur mörkrum, se till att stänga mörkgardiner efter sig; stäng av eller skym natriumlampan under torktid.              Överbliven dipemulsion hälls över i plaströr, vilka i sin tur läggs in i blycylindrar- märk dessa med   datum, samt om emusionen dippats i eller ej.

c.)  Packning- Under tiden som glasen torkar förbered boxar i vilka glasen skall exponeras- lägg ett tunt lager med 1-3 mm blågel i botten, täck med filterpapper (OBS-blågel absorberar fukt och skall tas “färsk” ur värmeskåp- blå=torr, vit=fuktig); märk boxar med relevanta uppgifter- dipdatum, diptid, box#, vävnad, subst, namn.
Då glas torkat, gå in i mörkrum under iakttagande av strikt mörkerdisciplin. Överför de torkade glasen till relevanta boxar; då alla glas är iplockade och locken till boxarna påsatta tejpas boxen runt om för att minimera risken för ljusinsläpp. De fyllda boxarna läggs sedan i blylådor för transport till och lagring i kylskåp vid 4°C i ISH/EM-lab.