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! |
