Is there anyone doing fat staining with C elegans?
I am doing Sudan Black B staining on well-fed and starved worms(both are wild-type), respectively. The results of the staining of those two kinds of worms show no difference(which is different from expected). I have several questions about this.
How long usually do I need to starve the worms in order to make them consume most of the fat stored in the intenstinal gut granules? I usually starve them for about 8~10 hours. I am not sure whether the time is not long enough, because when I look at the fixed starved worms under microscope, I can still see some granule-like thing inside their body.
I usually let the fixed worms stay in Sudan Black B staining solution overnight, which is suggested by the protocol. When I observe the worms under the microscope, I can see the whole body of worms were stained into some dark color, and the gut granules in the intestine, which should have dark-blue color, do not have the color that they are supposed to have.
When fixing worms for Sudan Black B staining, does it matter that I use 10% formaldehyde insteaded of 10% paraformaldehyde?
sudan black b staining is quite a tricky technique. in our lab we use fresh 2% paraformaldehyde to fix the worms, then we leave the stain overnight, after the overnight incubation, worms are destained with 70% ethanol. after destaining, you could easily see the contrast of the body (which is supposed to be unstained) and the intestinal gut granules (which should be dark blue to black). a possible alternative to staining your fat would be to try to soak your worms in a fluorescent dye (nile red). although i have no idea if nile red can actually distinguish a slight decrease in fat staining as that with sudan black.
good luck in your staining.
I have another question: in my experiment, I used both sudan black and nile red to stain the worms. nile red is working now, but the weird thing that I have now is that, even after I starved some of the worms(on an unseeded plates with nile red) for three days, when I observed them under fluorescence microscope, I can still see the gut granules(spots with nile red which show bright red color). That’s really weird, right? some one told me after starve the worms for 8~12 hrs, they should start mobilizing their fat storage and there should be nothing inside their intestine.
do you need to starve your worms in your experiment? When you centrifuge the worms, what rpm do you use? 2000 rpm for 1 min or 3000 rpm for 1 min, or it does not matter? Do you know how to get rid of the bacteria from worms. If the rpm is too high or the time is too long, will the bacteria also pellet with worms down the bottom?