Hello community,
Could anyone give me some tips about fixation conditions for staining worms with Sudan black?
Many thanks!
Alexandre ;D
Here’s the Sudan Black B staining protocol that I received from the Ruvkun lab some time ago; it worked well for me.
- Fixation
-Wash animals from unstarved plate(s) with M9 or PBS buffer. Wash worms in a 15 mL conical tube 2-3 times to get rid of bacteria. Incubate worms in 5-6 mL of the same buffer for another 30 minutes to excrete the bacteria in the gut, if desirable. Transfer worms to a microfuge tube, leaving them on ice.
-Add 10% paraformaldehyde (PF) stock to final concentration of 1%, mix well, freeze in dry ice/ethanol. Frozen samples may be processed immediately or stored indefinitely at -80C and processed as below.
[Note: 10% PF stock is suggested to be made fresh at least once a week by dissolving 1 gram of PF in 10 mL hot water (preheated in microwave oven), add 1-2 drops of 2N NaOH and incubate at 55-60C for 15 minutes.]
Melt under a stream of warm water until the ice is slurry, then freeze again, and melt again.
Repeat for a total of 3x freeze-thaw cycles.
After the final thaw, incubate samples on ice with occasional agitation for 10 minutes.
- Staining:
-Wash fixed worms in cold M9 or PBS at least 3 times.
-Dehydrate worms through an ethanol series: 25%, 50%, 70%. Keep worms in each gradient for 2 minutes sequentially. Aspirate ethanol.
-Add 2-5 volumes of Sudan Black B solution to the worms.
Solution:
Sudan Black B solution
A saturated solution of Sudan Black B (Sigma #S2380) in 70% ethanol.
This solution can be kept for prolonged periods. Filter with syringe and 0.45 micron filter before using.
Good luck!
Harold
Dear Harold,
Thanks for your reply! I really appreciated it.
I still have one basic questions: after incubation with Sudan black, should I proceed with a rehydrate step or just wash with ethanol 70% before mounting worms?
In (Padmanabhan et al., 2009), they didn’t rehydrate but in (Gomez et al.,2006) they did. In (Kimura et al.,) they didn’t depict this issue…
Alexandre ;D
I typically rehydrate the worms after staining, but I don’t think it makes a significant difference.
-Harold