RNA FISH
(on the basis of Edith Heard protocol) FIXING THE CELLS - Grow cells on slides (e.g. Superfrost Plus autoclaved glass slides - treated with 0.1% gelatin if cells have problems adhering- or plastic LabTek chamber slides from Nunc). OR
- Cytospin the cells onto a slide
- Rinse slides in 1XPBS at RT in baked coplin jar
- Incubate slides (permeabilise cells) for 4-7 (longer if needed, 15-30) min in CSK buffer + detergent ON ICE
- Fix slides in 4% paraformaldehyde/1xPBS, 10 min ON ICE
- Wash slides twice in 70% ethanol
- Store in 70% ethanol (at 4°C or -20°C for up to a month, We do not store for too long)
Cytoskeletal (CSK) Buffer: | Final conc. | Reagent | For 500 mL | For 1 L | MW
| 100mM | NaCl | 2.92g | 5.85g | 58.5 | 300 mM | sucrose | 51.3g | 102.69g | 342.3 | 3 mM | MgCl2 | 0.3g | 0.6g | 203.3 | 10 mM | PIPES | 1.5g | 3g | 302.4 | | Sterile H2O | to 500 mL | To 1 L | | - Adjust pH to 6.8 with NaOH if necessary
- Filter sterilise and store in aliquots at -20°C
- Prior to use add (to 50 ml aliquote):
Final conc. | Reagent | For 50ml | Notes
| 0.5% | Triton X100
(Sigma T8787) | 250 ml of 100%
(or 2.5ml of 10%) | dissolve by stirring -takes a while! | 1mM | EGTA | 250 ml
(from 200mM stock) | | 2mM | Vanadyl Ribonucleoside | 500 ml
(200 mM stock, Biolabs ref 1402) | | Fixative 16% Paraformaldehyde solution, EM Grade (EMS cat #15710) diluted to 4% in PBS PROBE LABELLING: We use the Nick Translation Kit from Vysis (Cat 32-801300) and label the DNA directly with SpectrumRed or SpectrumGreen. Alternatively, we use Biotin-Nick Translation mix (Roche, Cat.No. 1 745 824) and DIG-Nick Translation mix (Roche, Cat.No. 1 745 816). We purify the labelled probe on MicroSpin S-300 HR columns (Pharmacia, Cat. No.27-5130-01 – the same as we use for cleaning radiolabelled probes). DAY 1 PROBE PREPARATION : per slide:
50 ng probe
1 ml salmon sperm DNA (10mg/ml, Boehringer MB grade)
5 ml Cot-1 DNA* (Gibco BRL)
*if cDNA probe, none needed; if genomic or lambda probe can use less eg 2 ml. - Add 2 vol of 100% ethanol and dry in speed vac completely
- Resuspend in 5 ml Formamide, 37°C for 10 min
- Denature at 74°C for 7 min
- Add 5ml 2xHybridisation buffer and mix well
-  Keep probe on ice until needed
[if added Cot-1 DNA, leave probe at 37°C for 15-30 min for competition] 2X hybridisation buffer: | Reagent | 1 mL | 100 mL | 200 mL | 300 mL
| 20xSSC | 200 ml | 20 ml | 40 ml | 60 ml | 50% dextran sulphate | 400 ml | 40 ml | 80 ml | 120 ml | BSA (Biolabs) | 200 ml | 20 ml | 40 ml | 60 ml | Vanadyl Ribonucleoside | 100 ml | 10 ml | 20 ml | 30 ml | H2O | 100 ml | 10 ml | 20 ml | 30 ml | SLIDE PREPARATION - Dehydrate slides through 80%, 95%, 100% ethanol, 3 min each
- Air dry on 42 °C heating plate
- Add 10 ml of probe under 22x22 mm coverslip
(if cells are "mountainous" eg attached embryoid bodies, need to use twice as much probe - at same conc.!!!) - Seal the coverslips with rubber cement (Fixogum, cat# FIXO0050), put slides in a humidified chamber, cover with foil and incubate in a water bath or oven o/n at 37°C
DAY 2 WASHING - Pre-heat waterbath with coplin jars and solutions to 42°C, but agitate slides at RT
- Wash in 2xSSC/50% formamide* 3 x 5 min each
Cover slips should fall off in 1st wash - be careful not to scratch slide *eg. 50ml of 2xSSC and 50 ml of 100% formamide
pH 7.2-7.4 very important - check just before using - Wash in 2xSSC (pH 7) 3 x 5 min each
- Transfer to a jar with 4xSSC/0.1%Tween (Tween20 Sigma P9416)
- Mount in antifade with DAPI if detection is not needed. DONE!
DETECTION Skip antibody labelling if using direct-labelled probes. - Make antibody dilutions for detection of biotin and digoxygenin in detection buffer; if detect both at the same time make sure that antibodies are not cross-reacting
- Mix well and spin down at 14000rpm for 10-15 min at RT; keep ABs at RT in the dark before use
- Shake off excess fluid from the slide but never allow to dry;
- Incubate slide in 40 ml blocking buffer (also spun down) under a 24x40 mm coverslip, 30 min 37°C, humid chamber
- Shake off coverslip and incubate in 40 ml 1st antibody 30 min 37°C, humid chamber
- Wash 3 x 4xSSC/0.1%TWEEN with agitation at 37°C
- Repeat the procedure with all following antibody layers
- After last antibody, wash 3 x 4xSSC/0.1%TWEEN with agitation at 37°C
- mount in 10-15 ml Vectashield (antifade mounting medium, Vector labs, H-1000), containing 0.1 mg/ml DAPI (from a 1000 x stock of DAPI already in vectashield at 0.1 mg/ml) and cover with a 22x22 mm coverslip (squash out excess+bubbles).
Eg. Antibody layers for simultaneous biotin and digoxygenin detection Probe | Layer 1 | Layer 2 | Layer 3 | Layer 4 | Layer 5 | Biotin | BB | - | AV-TR | BAA | AV-TR | Digoxygenin | | AD-FITC | FITC-AS | - | - | Dilutions:
AD-FITC (anti-dig FITC, made in sheep)          1:15
FITC-AS (FITC-anti-sheep, made in rabbit)       1:100
AV-TR (avidin-texas red)                                 1:500
BAA (biotinilated anti-avidin, made in goat)        1:100 Blocking Buffer Reagent | 1ml | 400 ml | 600 ml | 800 ml
| 20xSSC | 200 ml | 80 ml | 120 ml | 160 ml | 10% TWEEN | 10 ml | 4 ml | 6 ml | 8 ml | BSA | 400 ml | 160 ml | 240 ml | 320 ml | H20 | 390 ml | 156 ml | 234 ml | 312 ml | Detection Buffer Reagent | 1ml | 2ml | 3ml | 4ml
| 20xSSC | 200 ml | 400 ml | 600 ml | 800 ml | 10% TWEEN | 10 ml | 20 ml | 30 ml | 40 ml | BSA | 100 ml | 200 ml | 300 ml | 400 ml | H20 | 690 ml | 1380 ml | 2070 ml | 2760 ml | Good luck! |
