Location: T-lab, level10, Microscope Room, Room7

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Lasers

Wavelength (nm)	Maximum power (mW)	Laser type
405	50mW
488	100mW
561	100mW
642	100mW

1. Huge data size/data handling(10Gb for 10 mins data collection)

2. Not enough fluorescence filter options for viewing under eyepiece(Only all color currently)

Dyes/Fluorophores….

1.MD/SC book, QP 519.9 mol. Pr 2008

Trimethoprim Derivatives for Labeling Dihydrofolate Reductase Fusion Protein in Living Mammalian Cells.

(Info on MS MLRK-DHFR construct, and TMP staining)

PALM Image of Dronpa-Paxilin, Focal Adhesion Complex, C2C12

6. Please see attached zeiss-elyra-quick-start-guide-pdf-cropped.pdf for the various settings


Sample Preparation for PALM

Only Photoactivatable/Photoswitchable/Photoconvertible ones can be used for PALM

Constructs:

Dronpa-paxilin(working)

Dronpa-actinin(? couldn’t expand)

tdEOS-paxilin(working)

tdEOS-vinculin(?couldn’t expand)

PA-GFP-tubulin(? low expression vector for speckle microscopy use)

Dyes:

TMP-hexachlorofluorescein(no lasers on current system)

TMP-uncaging rhodamine(can be uncaged, but DHFR transfection/dye staining need to be improved)

Items to be ordered:

Glucose Oxidase from Aspergillus niger Sigma G0543-50KU
Catalase from Bovine liver Sigma C3155-50 MG
Cysteamine = MEA 10 g Fluka 30070
Gold Colloid 80 nm (GM.GC 80) and 100 nm (GM.GC 100) from BB International (www.british-biocell.co.uk)

Problems with Sample preparation:

1. DHFR-TMP staining(current construct MLRK-DHFR, actin-DHFR)

2. redox cocktail for dSTORM

PALM type experiments with standard fluorescent dyes, such as Cy5 or Alexa 647.

1. Grow cells on glass(coverslips 25mm/Iwaki/Labtek chambers)prepared for TIRF according to your needs(coat with 0.01% Poly-L-Lysin or fibronectin for adhesion of the gold beads as described in step4) Make sure that the volume is small by using LabTek chambers or similar products.(No. 1/1.5 for thickness)

2. Wash cells twice briefly with 37 degree PBS.

3. Fix in 4 percent PFA for max. 10 minutes at room temperature to preserve some mobility for switching.Please warm up PFA to 37 degree as cold PFA tends to destruct microtubule integrity.

4. Wash cells twice briefly with PBS.

5. Optional: Permeabilize cells for 10 min, with 0.5 percent Triton-X-100 in PBS.

6. Block cells with 5% goat serum(sigma) in PBS for 15 mins.

7.Stain F-actin as follows:
a. Incubate cells with phalloidin-alexa 647(invitrogen/molecular probes,1~0.1 uM) in PBS for 1 hour.(actual staining concentration 6.6uM stock used at 1:100).

b. wash cells 3x in PBS supplemented with 0.1% Tween20(Sigma).

Stain microtubules as follows:
a. Incubate cells with an anti-alpha tubulin mouse monoclonal antibody(1:200/1:300)(invitrogen) in PBS for 1 hour.

b. wash cells with 3X in PBS supplemented with 0.1% tween 20(sigma)

c.Incubate cells with a goat anti-mouse F9ab)2 fragment conjugated with Alexa Flour 633, goat anti mouse(1:100) in PBS for 1 hour RT.

d. Wash cells 3 times in PBS supplemented with 0.1%Tween20(sigma)

8. Add gold beads(100nm, BI 1:100 diluted in PBS) and incubate for 2 hour(or longer). One should aim for 3-5 gold beads per field of view for recording to allow drift correction.

9.Wash cells 2 times with PBS.

10. Leave coverslip or dishes in PBS until recording and store at 4 degree. you might want to cover the chamber with paraFilm to keep the oxygen level low.
11. Add the following mix just before the experiment:
0.5ug/ml glucoseoxidase ; 40ug/ML catalast ; 10% w/v glucose ; 50mM 2-aminoethanethiol(trivial name beta-mecaptoethylamine or cysteamine)

(actual used, 1xPBS, 10% final volume of glucose, 40ug/ml catalase,500ug/ml oxidase,50mM of cystamine) – basically, home made anti-fade agent for TIRF samples but it doesn’t work well under our hands… sample still get bleached pretty fast
12.Perform PALM experiment. Set the laser power(640nm) to about maximum, after initial bleaching. Recording needs only mild activation(488nm) after some minutes.
13.After microscopy, wash cells 3 times in PBS and store at 4 degree under PBS. Seal with Parafilm to avoid evaporation.

PS: One might wish to starve cells(0.5% serum, overnight(15 to 24 hours) followed by stimulation with growth factors to get better stress fiber.

=.=