Biology Reference
In-Depth Information
- 5 min incubation in 0.5% Triton X in MTSB
- 15 min incubation in 4% PFA in MTSB
- 5 min wash in PBS
- 5 min incubation in 100 mM glycine in PBS (in order to quench remaining
aldehyde groups)
- 5 min wash in PBS at RT, followed by an optional storage at 4 C in PBS.
All the solutions should be at RT in order to preserve intact MTs inside the cells (in
all steps prior to the PFA fixation, MTs depolymerize if cold solutions are used).
16.2.3 Immunocytochemistry on fixed cells
Coverslips with fixed cells were subjected to indirect immunochemistry. Antibodies
have been diluted to the desired concentration in PBS, 3% bovine serum albumin,
0.1% Triton X-100 (this solution can be filtered, aliquoted, and stored at
20 C).
In order to use a minimum amount of antibodies, we incubate coverslips with at-
tached cells facing downward on a drop of 30 m l of antibody solution in a humid
chamber for 1 h at RT. 12G10 is a monoclonal antibody detecting a -tubulin
(Developmental Studies Hybridoma Bank, University of Iowa). C105 is a rabbit
polyclonal, anti- b -tubulin antibody (gift of Jose M. Andreu, Madrid; Arevalo,
Nieto, Andreu, & Andreu, 1990 ). The coverslips were then transferred to a 24-well
plate and rinsed three times with PBS, 0.1% Triton X-100, and subsequently
incubated with the fluorescent secondary antibodies as previously described (Life
technologies #A11001 Alexa 488 anti-mouse IgG, #A11019 Alexa 568 anti-mouse
IgG, #A11036 Alexa 568 anti-rabbit IgG, all used at 2 m g/ml, and #A21068 for Alexa
350 anti-rabbit IgG used at 10 m g/ml). The coverslips were again transferred to a
24-well plate and rinsed four times with PBS, 0.1% Triton X-100 (if DNA staining
was required, the first wash was performed in PBS, 0.1% Triton X-100, 0.1 m g/ml
DAPI for 3 min at RT). Coverslips were then mounted carefully on microscopy
slides using 6 m l of Mowiol solution. Mowiol is left to polymerize by leaving the
slides in horizontal position at RT and in the dark for at least 4 h, and most commonly
overnight.
16.2.4 Microscopy and image acquisition
Images were acquired on a Leica DMRXA upright microscope using Metamorph
(Molecular Devices) software. Images were treated using ImageJ ( http://imagej.
nih.gov/ij/ ) or Photoshop (Adobe Systems).
16.2.5 Electrophoresis and immunoblot
We use a variation of SDS-PAGE allowing for the separation of a - and b -tubulins as
two distinct bands. The method has been described in detail in Lacroix and Janke
(2011) . It differs from the standard SDS-PAGE protocol by a different acrylamide
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