3.12@ Observation of Microtubule with indirect
immunofluorescence microscopy
2xPME
|
|
Final conc. |
0.5 M
PIPES-NaOH (pH6.8) |
120 ml |
0.2 M |
0.5 M EGTA
(pH8.0) |
3 ml |
5 mM |
1 M MgCl2 |
0.6 ml |
2 mM |
H2O |
176.4 ml |
|
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ @@ 300 ml@@@@
*0.5 M PIPES-NaOH
500ml requires ca. 14 g NaOH.
10% NP-40
Stock
solution |
1 ml |
Final conc. |
PMSF (wako) |
17.4 mg |
100 mM |
2-prophanol |
1 ml |
|
Store
at –20˚C.
Fixation
solution
Stock
solution |
10 ml |
Final conc. |
Formalin |
2.16 ml |
8% |
2x PME |
5 ml |
1x |
10% NP-40 |
10 µl |
0.01% |
DMSO |
100 µl |
1% |
100 mM
PMSF* |
50 µl |
0.5 mM |
H2O |
2.68 ml |
|
Make new
fixation solution every time as needed.
*Optional
PMEN0.01
Stock
solution |
500 ml |
Final conc. |
2x PME |
250 ml |
1x |
10% NP-40 |
0.2 ml |
0.01% |
H2O |
250 ml |
|
10%
Triton-X 100
Stock
solution |
1 ml |
Final conc. |
Driselase
mix |
1 ml |
- |
25x
Proteinase inhibitor (BM) |
40 µl |
1 x |
*Driselase
mix (stored at –20˚C)
Stock solution |
10 ml |
Final conc. |
Driselase
(Kyowa) |
0.2 g |
2% |
0.5 M EGTA
(pH8.0) |
100 µl |
5 mM |
8% mannitol |
6.25 ml |
5% |
H2O |
Fill up to 10 ml |
|
Mix well,
centrifuge, and filtrate with 0.22 µm filter.
Stock
solution |
1000 ml |
Final conc. |
NaCl |
8 g |
137 mM |
Na2HPO4
7H2O |
2.2 g |
8.1 mM |
KCl |
0.2 g |
2.68 mM |
KH2PO4 |
0.2 g |
1.47 mM |
H2O |
1000 ml |
|
For
immobilizing specimens to a coverslip, PEI is used.@
Drop 3
µl of 0.1% PEI to a coverslip and put another coverslip on it. Spread out
the solution over the both surface of the coverslips. Dry up at room temperature.
Store at -20C.
Stock
solution |
10 ml |
Final conc. |
10% Triton
X-100 |
1 ml |
1 % |
BSA |
0.1 g |
1 % |
Store
at –20˚C.
0.2
µg/ml DAPI solution
Dilute to 2
mg/ml with PBS when use.
Procedure
1. Pour the
fixation solution to a 3.5 cm petri dish. Put a small colony of protonemata
(about 3 mm in diameter) to the fixative. Shake gentry for sinking down the
specimens and incubate at room temperature for 60 min.
2. Rinse with PMEN0.01
(10 min x3)
3. Put the
specimens on a PEI-coated coverslip and remove the excess solution with a filter paper.@ Drop a PMEN 0.01 and confirm the attachment on the coverslip.
4. Drop 5-10
µl driserase solution (10 min at room temperature).
5. Rinse with PMEN0.01
(10 min x3).
6. Remove excess
water and drop cold methanol (10 min at -20˚C).
7. Rinse with PMEN0.01
(10 min x3).
8. Drop 10
µl of 1/20 diluted Triton/BSA (10-15 min at room temperature).
9. Rinse with
PBS (x3).
10. Incubate with
primary antibody at 4˚C overnight or 2-4 h at 37˚C.
11. Drop 2-5
µl of 1/100 diluted mouse a-tubulin monoclonal antibody (Oncogene, clone
DM1A, cat# CP06) to the specimens.
12. Rinse with
PBS (10 min x3).
13. Incubation
with the secondary antibody for 1-3 h at 37˚C.
14. Drop 5
µl of 1/500 diluted Goat anti-mouse IgG antibody (Molecular Prove, Alexa 488-cat.
No. A11001, Alexa 546-cat no. A11003).
Keep samples
in a dark box during the following procedures.
15. Rinse with
PBS (10 min x3).
16. Drop 5
µl of 0.2 µg/ml DAPI (10 min at room temperature).
17. Rinse with
PBS.
18. Mounting with
PBS and sealing with rubber cement.