3.8@ Observation of microtubules with indirect immunofluorescence microscopy
In
this section we describe a protocol for antibody labeling of protonemata.
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-propanol |
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 filter 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 on a coverslip, PEI is used.@
Drop
3 µl of 0.1% PEI on a coverslip and put another coverslip on it. Spread
out the solution over the surface of both coverslips. Dry 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 into a 3.5 cm petri dish. Put a small colony of protonemata
(about 3 mm in diameter) in the fixative. Shake gently to sink 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.@ Add a drop of
PMEN 0.01 and confirm
their attachment on the coverslip.
4. Drop 5-10
µl driselase solution (10 min at room temperature).
5. Rinse with
PMEN0.01 (10 min x3).
6. Remove
excess water and add a drop of 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 room temperature.
11. Drop 2-5
µl of 1/100 diluted mouse anti-tubulin monoclonal antibody (Oncogene,
clone DM1A, cat# CP06) onto the specimens.
12. Rinse with
PBS (10 min x3).
13. Incubation
with the secondary antibody for 1-3 h at room temperature.
14. Drop 5
µl of 1/500 diluted Goat anti-mouse IgG antibody (Molecular Probes, 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. Mount with
antifading reagent and seal with rubber cement.