Help with unwanted fluorescent lines in sections
Posted 13 June 2009 - 09:27 PM
Posted 15 June 2009 - 08:00 AM
you sure it isnt positive staining?
Posted 15 June 2009 - 06:11 PM
And you used a mouse antibody on mouse brain? did you use something like a mouse-on mouse kit to reduce cross-reactivity or anything?
Posted 15 June 2009 - 10:07 PM
Posted 15 June 2009 - 10:36 PM
Did you do a secondary antibody control slide? No primary just the secondary to see if that reacts with anything?
Posted 16 June 2009 - 06:06 AM
Posted 16 June 2009 - 04:21 PM
Usually you shouldn't get any meaningful staining from secondary controls (non would be beautiful). I think your big problem is using the mouse antibodies, because your tissue is mouse then its going to be hard to tell what yr antibodies are really staining. It sounds like your primary is picking up something, Is it a well characterised antibody- as in lots of references on the data sheet you can check their staining against?
If you have another species antibody you can use- say Rabbit polyclonal what does the staining look like for that.
Posted 16 June 2009 - 04:23 PM
Posted 16 June 2009 - 04:32 PM
Just an aside question for anyone wishes to answer. Can a different secondary antibody play a big role in staining? For example, I usually use Cy2, but the staining is ok, not awesome, sometimes good, other times not. So maybe it's my primary or something wrong with my slices (ie. not perfused properly maybe???). Anyhow, today I tried for the first time Alexa 488, and lo and behold there were lots of immunoreactivity that I didn't see with Cy2.
Essentially they are the same anti rabbit or anti mouse, no?
Posted 16 June 2009 - 04:57 PM
My labs have used the mouse on mouse kit from vector labs, they're very good.
Anyway, I hope it helps,
Posted 16 June 2009 - 05:05 PM
Posted 16 June 2009 - 06:20 PM
Posted 17 June 2009 - 01:01 AM
then this means what you think is positive staining is actually background - and those lines are probably the real staining - sorry - looks like you were looking at the wrong bit
an alternative to mouse on mouse kit is just use some unlabelled anti mouse secondary eg a fluorochrome well away from the one you are looking at or biotinilated anti mouse (as long as you arnt avidin/biotin-ing currently)
ahh - gotta live science - never works as you expected - but keeps you on your toes