endothelial cells' staining - (Nov/28/2005 )
Dear all,
I have been trying to characterize endothelial cells that were extracted from the aorta by a lab member.I have been having problems doing the same as even my negative control (one w/o primary Ab) also shows a good signal..pls help!Also,if u could suggest any other fibroblast specific marker besides vimentin it'll be great
I have been trying to characterize endothelial cells that were extracted from the aorta by a lab member.I have been having problems doing the same as even my negative control (one w/o primary Ab) also shows a good signal..pls help!Also,if u could suggest any other fibroblast specific marker besides vimentin it'll be great
What animal is the endothelial cell from?
did you considere using LDL uptake?
LDL uptake didnt quite work for me...used NIH 3T3 as controls which also ended up taking LDL!!!
Also: Do you know if endothelial cells change morphology (appear spindle like) when they are motile??
Yes knowing what animal the cells are from will help. What antibodies have you tried and what was your protocol. I can help.
What animal is the endothelial cell from?
My cells have been extracted using the non-enzymatic method from the aorta of a mouse.I;ve been tryint o stain for CD31(PECAM). i fix the cells in acetone for 10mins,followed by quick washes,blocking in goat serum,incubation with rat anti mouse CD31 (1:100) at 4C o/N-----washes in PBS,incubation with biotinylated goat anti-rat for 30 mins,washes---incubation with alexa 488---washes----dapi--mount.
all washes are in PBS.
my negative cntrl--cells w/o primary and only secondary shows staining too.........is secondary a problem----(but it works for embryo staining)---is fixation a problem----------or are cells a problem (do they need to be at hisher confuence)????????
pls help!!!!!!!!!!
What company is the CD31 from? What clone? Is it MEC13.3? If so, it should and will work! Don't worry!! It's an AWESOME antibody if the conditions are right.
A few questions:
- Are you sure your cells that you extracted are endothelial cells and not fibroblasts? Do you have a positive control for your stain, something you KNOW expresses CD31 to make sure all reagents are good and functional?
- Use the antibody at 1-5 ug/ml. You say a dilution of 1:100 you are using but what is the concentration?
- I also use acetone (5 min is sufficient) so I don't think that is the problem but why not also try 1% PFA/PBS for 15 minutes as a fix just in case (normally this antibody doesn't stain well with PFA but it's worth a shot).
- The background you see may be endogenous biotin, do you block it?
- Do you examine the slides immediately after staining to make sure nothing is happening over time between the stain and when you examine the slides?
- Confluence isn't an issue I wouldn't worry about it.
You need to use CD31. It's a perfect marker for EC.
Good luck,
Alex