In-situ protocol : Antonio, 2001
Day 1
- Adjust oven to 37 oC : during the permeabilization & prehybridization steps, then 53oC during hybridization (O/N)
- Adjust oven to 42 C for washing step in the next day and put reagents in
the oven O/N
PCR for RLP from Abalone
Reagents 50 ml reaction (recommend 25 ul Rxn)
sdw 24.6 mlMgCl 1.5
Buffer 5
TMAC 5
DNTPs 4
BSA 10 mg/ml 0.5
Primers
RA5-1 2
RA3-6 2
Taq 0.2
DNA template 3
Cycling conditions
1 = 95°C for 5:002 = 94°C for 1:00
3 = 62°C for 0:30
4 = 72°C for 0:30
5 = Go to 2, 39 times
6 = 72°C for 7:00
7 = 16°C forever
Labeling by PCR using PCR DIG Probe Synthesis Kit
Roche Applied Science: Cat No. 11 636 090 910Add the following components to a sterile microfuge tube on ice:
Reagent Volume (50ul reaction) (recommend 25ul rxn)
sdw 29.25
PCR buffer (vial 3) 5
PCR DIG mix (vial 2) 5 : Use DNTP in kit for negative control
Primers (upstream, downstream)
RA 5-1 (20 pml/ml) 2.5
RA 3-6 (20 pml/ml) 2.5
Enzyme mix (vial 1) 0.75
Template DNA
(1:100 dilution of RLP plasmid) 5
Note: Conditions for labeling are the same as conventional PCR outline above. Always include one reaction without DIG labeling (i.e. regular PCR reaction containing RLP DNA template).
Important: Before using the DIG labeled probe, denature the probe at 95C for 3 minutes and immediately place on ice for about 30 min to separate the double stranded DNA. Store at –20 or –70C until use.
Tissue Deparaffinization ~ 1 hr 20 min
1. Deparaffinize with 3 changes of safeclear (xylene) for 10 min each.
2. Hydrate with graded ethanol series of (100,100, 95, 80, 70, and 50%), for 3 min
each.
3. Then rinse slides in sterile dH20.
4. Equilibrate tissue sections in Tris buffer pH 7.2 for 5 min
0.2 M Tris-HCl
2.0 mM CaCl
Permeabilization of tissues ~ 1 hr 20 min
1. Add tissues with 50 ug/ml Proteinase K in Tris-buffer for 45 min at 37 oC.
2. Rinse tissues with PBS: 3x10 min.
Prehybridization ~ 2 hr
1. Prepare prehybridization buffer (Total volume = 1 mL):
0.51 mL deionized formamide
0.20 mL 20x SSC
0.05 mL heat-denatured sperm DNA (10 mg/mL)
0.20 mL 50% Dextran Sulfate
0.02 mL 50x Denhardt’s
USE THIS: 1.1 Alternative prehybridization buffer (4xSSC ; 50% deionized formamide) ;
Example. we need 10 ml prehyb.buffer
deionized formamide = 5 ml
20xSSC(4xSSC) = 2 ml
Add DEPC H2O = 3 ml
2. Incubate in in prehybridization buffer for 1-1.5 hr at 37C OR 2 hr at
RT in humid chamber.(Alternative incubate at least 10 min)
Hybridization ~ 30 min.
1. Discard prehybridization buffer and rinse slides in 2x SSC and briefly dry prior to
hybridization.
2. Add probe to prehyb. buffer at a concentration of 1:373
3. Add probe to tissue sections. [300 ul each]
4. Cover tissue sections with Dnase-free coverslips.
5. Incubate slides at 53C O/N in a humid chamber.
Stringency washes Day 2: Carefully remove cover slips from sections by immersing slides for 5-10 min in 2xSSC at RT
1. Wash slides in 2x SSC twice for 15 min each at 40 oC.
2. Wash in 1x SSC three times for 15 min at 40 oC.
3. Wash in 0.5 x SSC for 15 min at 40 oC
4. Equilibrate tissues in Buffer 1 @ RT (100 mM tris-HCl, 10 mM NaCl, pH 7.5) for 10 min
5. Block tissues with Blocking Buffer (Buffer 1 + 2% sheep serum + 0.3% Triton X-
100) for 1 hr at RT. {500ul each}
6. Never let the sections dry out.
Detection
1. Prepare Alkaline phosphatase (AP)-labeled sheep anti-DIG antibody conjugate. Dilute
antibody 1:1000 in Buffer 1 + 1% Sheep serum + 0.3% Triton X-100.
2. Add solution (0.5 -1 mL) to dry section and incubate without coverslip at RT
For 2 hours in a humid chamber.
3. Rinse slides with Buffer 1 for 2 x 10 min.
4. Rinse slides in Buffer 2 (100 mM tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5 –
make sure!!) for 10 min.
5. Prepare substrate by adding 45 ul NBT (nitroblue tetrazolium) + 35 ul BCIP (both light sensitive) to 10 mL of Buffer2
6. Incubate tissue sections with substrate solution 30 min -1 hour at RT in humid chamber. Check color after 15min. If not developed, incubate O/N.
7. Rinse slides in 3x dH2O OR dip slide breifly
8. Counter stain slides with 0.05% aqueous Bismarck Brown Y for 3 min.
9. Rinse slides with dH2O, then 70% and 100% EtOH breifly OR just running tap water for 10min
10.Let slides air dry then permount.
Note:
To optimize the protocol the following variables can be modified:
1. Concentration and length of solubilization with proteinase K.
2. Dilution of probe and anti-DIG-AP antibody conjugate
3. Temperature and length of incubation of anti-DIG antibody
4. Temperature and stringency of washes
5. Length of incubation of BCIP-NBT substrate. Signals should develop beginning at 30 min to 1 h. Do not incubate beyond 1 h.
Solutions
Tris Buffer
0.2 M Tris-HCl
2 mM CaCl
pH 7..2
PBS pH 7.4
1x SSC, pH 7.0 2x SSC, pH 7.2 20x SSC-store at 20C
0.15 M NaCl 0.3 M NaCl 3M NaCl
0.015 M sodium citrate 0.03 M sodium citrate 0.3M Na citrate
pH 7.0 pH 7.2 DI H2O
pH 7.2
Prehybridization Buffer
0.51 mL deionized formamide
0.2 mL 20x SSC
0.05 mL heat-denatured sperm DNA (at 10 mg/mL)
0.2 mL 50% dextran sulfate
0.02 mL 50x Denhardt’s solution
Add Poly A at 0.1 mg/mL
Buffer 1 :
100 mM Tris-HCl ; Molar = g/MW; g = M x MW = 0.1M x 157.6 = 15.76g in 1 L H2O
10 mM NaCl ; 0.01 x 58.44 = 0.5844 g in 1 L H2O
pH 7.5
Blocking Buffer
2% Sheep serum
0.3% Triton X-100 in Buffer 1
Antibody Solution
Alkaline Phosphate (AP)-labeled sheep anti-DIG antibody conjugate diluted 1:1000 in
Buffer 1 with 1% sheep serum + 0.3% Triton X-100
Buffer 2
100 mM tris-HCl
100 mM NaCl
50 mM MgCl2
pH 9.5
Day I
I. Abalone Rickettsia Okigonucleotide Primers: Optimum oligonucleotide conc. = 200 pm/ul
Primer Conc (pmole/ul)
RA 5-1 234
Have 234 pmole/ul (X) = want 200 pmole/ul x 200 ul?
X = 170.94 ul
RA 3-6 183 = 218.57 ul
RA 3-8 151 = 264.90 ul
RA 5-6 238 = 168.067 ul
Total = 822.477 ul
Then:
RA 5-1: Have 822.477ul (X) = want 0.992 ul x 170.94ul
X = 0.206 ul
RA 3-6: = 0.264 ul
RA 3-8 = 0.319 ul
RA 5-6 = 0.203 ul
Total = 0.992ul
II. Control group: = 5 ul viral # 6 instead of the primers
4 ul Dnase free-water
Then add reagents (11 ul).
So the total of the Labeling procedure is 20 ul.
III. Permeabilization of tissues
- Wash tissues with 50 ug/ml of Proteinase K in Tris-buffer for 45 min at 37 C.
Working 50 ug/ml of Proteinase K in Tris-buffer.
Proteinase K: stock 20 mg/ml = 20,000 ug/ml
Example: Have 20 mg/ml (20,000 ug/ml) (X) want 50 ug/ml x 2,000 ul
X = 50 ug/ml x 2,000 ul/20,000 ug/ml
= 0.005 ml
Take Protein K = 5 ul
Tris buffer = 2,000 – 5 = 1,995 ul
We Use about 15 ml/4 slides
Then we want 15 ml of 50 ug/ml Proteinase- K in Tris-buffer
Have 20,000 ug/ml (X) want 50 ug/ml 15 ml
X = 50 ug/ml x 15,000ul /20,000 ug/ml
Take Protein K from stock = 37.5 ul
Tris-buffer =15,000 ul -37.5 ul = 14.97 ml
IV. Prehybridization buffer (Total volume = 1 ml/RXN),but we have to dilute the antibody in the prehybridization for the next step : so I would prepare for those.
Volume (ul)1 RXN reagents 3.5 RXN (7 samples)
510 ul deionized formamide 1,785 ul
200 ul 20X SSC 700 ul
50 ul heat-denatured sperm DNA (10 mg/mL) 175 ul
200 ul 50% Dextran Sulfate 700 ul
20 ul 50X Denhardt’s 70 ul
* For 50% Dextran Sulfate : powder (on the shelf)
Weigh 50 g in NanoH2O 100 ml
5 g in:::::::::::::::::::::10 ml
0.5 g in NanoH2O 1 ml
- I want to stock 10 ml of 50% Dextran Sulfate
Then stir for 3 min.
V. Hybridization
Probe : Prehybridization buffer = 1:373 (Dolly recommend: 13:4850 )
Day 2
VI.Stringency washesBlocking buffer (5 ml/ 10 RXN) – I think , blocking buffer need per slide = 500 ul.
100 ul 2% sheep serum
15 ul 0.3% TritonX-100
4,885ul Buffer 1
Total 5 ml
VII. Detection
Antibody Buffer (7 RXN) = 7 ml Buffer 1
= 21 ul 0.3% TritonX-100
= 70 ul 1% sheep serum
Dilute antibody 1: 1000 in Antibody buffer
Ex. 7 ul : 6,993 ul
VIII. Prepare substrate (ex.)
4 RXN 8 RXN
5 ml Buffer 2 10 ml
22.5 ul NBT 45 ul
17.5 ul BCIP 35 ul
IX. Counter stain : Stock : Bismarck Brown Y 25 g as powder
I want 0.05% Bismarck Brown Y
0.05 g in Nano H2O 100 ml
Weigh 0.05 g of Bismarck Brown Y.
Suggested to dilute Bismarck Brown Y
0.05% to 1:10 in PBS (Neutral pH)