Biology Reference
In-Depth Information
Tips
Depending on the cell type, adjust the amount of Triton X-100 in the
permeabilization buffer, between 0.03% and 1% so that cells remain attached
to their substratum. Cells indeed tend to detach very easily after Triton
permeabilization. All the steps performed prior to fixation must be made
cautiously. As much as possible, do not aspirate or flow medium over the
cells. Prefer holding the coverslips between tweezers and process by short
immersions.
10.1.2
MB11 staining
Immunofluorescence of GTP-tubulin should be performed in a wet chamber at
37
C. Drops of antibodies are deposited on a sheet of Parafilm in a closed and
humid chamber.
Both MB11 and the antihuman secondary antibody are diluted in PEM-G
supplemented with 2 g/L BSA.
After permeabilization, coverslips are put on a drop of MB11 antibody and
incubated for 15 min at 37
C. After incubation, prior to washes, gently add warm
PEM-G under the coverslips before picking them up to prevent cells from
detaching.
Wash out the primary antibody (3 times, 1 s) in a warm PEM-G bath.
Put the coverslips directly on the secondary antibody drop, on Parafilm, in
the humid chamber. Incubate 15 min at 37
C. Once again, lift slowly the
coverslips using PEM-G, wash with caution in PEM-G, and fix the cells.
10.1.3
Cell fixation and colabeling
Cells can then be fixed either with methanol or with paraformaldehyde (PFA)
depending on the needs of the experiments.
Fix the cells with cold methanol (
20
C or with 3-4% PFA
for 15 min at room temperature. Wash twice with PBS. Once the cells are fixed, a
classical immunofluorescence can be performed for colabeling GTP islands with
another staining. Note that cell permeabilization preserves the conformation of
microtubules and of tubulin, but plus ends-tracking proteins (
20
C) for 4 min at
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TIPs) are lost
from microtubule plus ends.
Tips
- After secondary antibody incubation and washes in PEM-G, put the
coverslip on a sheet of Whatman paper to eliminate residual PEM-G.
Directly immerse the coverslip in cold methanol.
- After the entire methanol is removed, quickly add PBS in one step. This
step is very important to keep cells in good shape and image individual
microtubules spread out in the cell.
