3.8@ Observation of microtubules with indirect immunofluorescence microscopy

 
Yoshikatsu Sato and Takashi Murata

 

 

In this section we describe a protocol for antibody labeling of protonemata.

 

 

Materials

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

 

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*0.5 M PIPES-NaOH 500ml requires ca. 14 g NaOH.

 

10% NP-40

 

100mM PMSF

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

 

Driselase solution

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.

 

PBS

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

 

 

PEI (polyethyleneimine)-coated coverslip

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.

 

Methanol

Store at -20C.

 

Triton/BSA

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.