Dil Sensory Neuron staining in nematodes

From Wikionchus

Jump to: navigation, search

The lipophilic dye Dil stains amphid neurons and phasmids


Staining

1 wash a plate of mixed stage worms with 10 ml of M9 to a 15ml Falcon tube centrifuge discard the supernatant and pipet the worms into 200µl of M9 in microcentrifuge tubes.

Total volume is now around 250µl

2 Add 1.5µl of Dil, mix well and incubate in darkness with shaking every hour for 3 hours at RT

3 wash worms. Pipet the worms on the bottom of new tubes filled with 800µl of M9. Centrifuge 1min at 2000rpm and discard the supernatant. Repeat that step twice.

Resuspend the worms in 500µl of M9

4 pipet the worms onto NGM agar plates seeded with OP50 and allow the worms to crawl in the OP50 spot for two hours to remove adhering dye at the surface.

5 pick 10-15 worms into M9 with azide to visualize the staining under Nomarsky microscope

6 since dauer larvae of Diplogastridae are covered with a lipophilic substance use 0.02% Triton-X in M9 to get rid of the oily substance


Imaging (on Zeiss Axioplan equipped with Metamorph software)

1 40x or 63x magnification setting. Auto exposure and take a picture to calculate the exposure time setting. Reset to "manual" after obtaining the exposure time and make sure it´s also set the same in "multidimensional aquisition"

2 keeping shutter open in "multidimensional aquisition"

3 close shutter to binoculars (on microscopy)to reduce extra light into camera. Set the right Z-stack value according to magnification (e.g. 0.267 for 63x) and set top and bottom plane to take images

4 crop and stack only the necessary area to minimize file size

5 preserve stack as 12-bit but do a maximium stack to see if one stacked image is good enough to get all patterns

6 using the off-line computer with Metamorph, do 3D reconstruct (maximum setting)

7 if there´s too much background light, process the image in Deblur/DeConvolution programm before 3D reconstruction

8 "8-bit sample" entire stack

9 "make movie" and save as Quicktime file (use 5 1/30 of a second per frame)

Links

Retrieved from "http://www.pristionchus.org/wiki/index.php/Dil_Sensory_Neuron_staining_in_nematodes"
Personal tools