Indirect Immunofluorescence

1. First antibody: anti-P-tubulin antibody (monoclonal from mouse; Sigma; see Table 1).

Table 1

Antibodies and Fluorescent Reagents Used in the Study

Table 1

Antibodies and Fluorescent Reagents Used in the Study

Reagent

Type

Species

Dilution

Fluorochrome

Max/Abs-em

Origin

Fluorescein-12-dUTP

dNTP

1/100

FITC

492/520 nm

Boeringer

Anti-b tubulin

IgG monoclonal

Mouse

1/40e

Sigma

Anti-Fc mouse

F(ab)'2

Donkey

1/50e

TRITC

550/570 nm

Jackson immunoresearch

TOTO3

Intercalant

1/1500e

642/660 nm

Molecular probes

DAPI

Intercalant

1/20,000

360/460 nm

Observation Systems Used For Acquisition With the Confocal Microscope

Table 2

Observation Systems Used For Acquisition With the Confocal Microscope

Laser beam

X

Fluorochrome

Dection filters

Blue argon

488 nm

FITC

BP 505-550

Green helium neon

543 nm

TRITC

BP 560-615

Red helium neon

633 nm

TOTO3

LP 650

2. Second antibody: tetramethylrhodamine isothiocyanate (TRITC)-labeled anti-mouse F(ab')2 fragments (from donkey) (Jackson Immunoresearch, West Grove, PA; see Table 1).

3. DNA counterstained once: TOTO 3-iodide (Molecular Probes, Paris, France; see Table 1).

4. 1 mMDimethyl sulfoxide (DMSO) (Sigma).

5. DNA counterstained twice: 4',6-diamino-2-phenyllindole (DAPI; Sigma; see Table 1).

6. Antifading: Vectashield anti-fading (Vector Laboratories, Burlingame, UK).

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