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Role of Numa in MT organisation? - Coggle Diagram
Role of Numa in MT organisation?
HR microscopy
Calibrated glass coverslip (1.5H)
Good SNR
Sample close to coverslip (ideally on coverslip)
Addapted fluorophores
SIM
All
STED
STAR RED STRAD Orange
To order
STROM
AF 647 AF555
AF555 not good on VUTARA, test CF 568
Addapted mounting medium
SIM
Prolong glass (solid)
STORM
Glox Abbelight Eternity
STED
Results
Apotome
Muscle slices
*
Confocal
Myotubes
*
*
SIM
Muscle slices
*
Isolated fibers
*
*
STED
on going
STORM
Isolated fibers
Tubulin tot
*
*
Tub tyr
*
Nikon AX
Muscle slices
Deconvolution
*
No deconvolution
*
Samples
Isolated fibers
Coverslip Coating with gelatin
Autofluorescence
Quenching Test with TueView
*
:check:
Primary culture
on treated plastic support in matrigel
Inclusion in OCT
:red_cross:
Inclusion in Histogel
:red_cross:
Plastic bottom cutout
with a laser
:red_cross:
with punch
*
SIM
*
:check:
Pay attention, coverslip must not be tilted
STED
:question:
STROM
:red_cross:
Direct observation
SIM
OK but not optimal
STED
:question:
STROM
:red_cross:
Try CI on glass coverslip, ibidi or Wilco
Need to be optimized
Muscle slices
Cryosection on Coverslip Coating with gelatin
*
don't work with heat Unmasking
Pericentrin labelling
:red_cross:
Try with enzymatique unmasking
Try an other coating
Hexamethyldisilazane (Jean Luc Thomas)
:check:
*
:check:
labelling without heat unmasking
Tubulin labelling don't work on frozen tissu
*
Try Vibratome section
Labelling of interest
*
Numa1
rabbit
*
Work with all fixation procedures tested
*
:check:
Good combination
Pericentrin
mouse IgG1
*
Work with special procedure
Primary culture
*
:check:
Muscle slices
*
:check:
Tubulin
D Tyr
rabbit (grenoble)
:check:
:no_entry: but not compatible with Numa labelling
mouse IgG2a
*
on going
Acetylated αTubulin
mouse IgG2b
*
*
on going
α 1a Total
mouse IgG1
*
:check:
:no_entry: but not compatible with pericentrin labelling
Tub Tyr
rat
:check:
the best labelling
AKAP 450
:red_cross:
reference present in the lab doesn't work
Buy and try an other reference
:warning: Numa colabelling need other than rabbit specie
CAMSAP2
rabbit
:check:
Tested by Léa ponctiform labelling in cytoplasm
*
:no_entry: but not compatible with Numa labelling
Others
Nedd1
γ tubulin
mouse IgG1
*
Myotubes (Léa)
:check:
HAUS complex
Dynein
DHPR
AcI directly labeled
Commercial
Pericentrin
Pericentrin 488 ref abcam 270118
*
Home made
