inversed microscope and fluorescent anti-CD34? - (Oct/03/2007 )

Does anyone know whether one can see fluorescent anti-CD34 antibodies on a "normal" inversed optical microscope? (up to 100x)

i dont know the specifics of your setup but to get fluorescence to work you need a wavelength to stimulate the fluorochrome at and another to observe it at - we use lasers to stimulate (can use filters - v good ones) and a confocal microscope to remove the laser wavelengh from what we see.

if you want to see your antibodies on a light microscope try a pap/dab label (ignore the fluor attached to your primary and use a secondary to the animal the primary was raised in) this will give you brown spots to look at (dont forget to counter stain - mayers haemalum would probably do)

dom

(by the way this isn't exactly text book - i dont know how a fluor attached to a primary will affect the secondary but i'm pretty optimistic - good luck)

Thanks for yours answer!

Yes, I know how to excite the fluorescent dye and observe the outcome fluorescence. We too, we use lasers.

We use BD anti-CD34 FITC which according to them can only be used for flow cytometry. So I wondered whether I could use it too to verify on an inversed microscope if everything works like it should.

the inversed bit is irrelevant - are you talking about light microscopy or fluorescant

if its fluor then fitc works just like oregon green or alexafluor488 - you will probably have to change the dilution from flow to immunohist but otherwise it should be ok - the company website will give you a better idea as they tend to specify the use of their antibodies

dom

Ok, many thanks to everyone.

I am in fact speaking of fluorescent microscopy, yes. I'm going to have a look at the BD website in oder to check the concentrations for using the antibody for immunostaining. The concentration for the flow cytometry is 3 to 60 microliters (antibody to PBS). Or do you have an idea of what concentration I should try?

if its tissue try 1:50, 1:100, 1:200, 1:500 that will allow you to optimise pretty well
we tend to put antibodies in bsa (bovine serum albumin) in pbs (0.2g in 10ml) - less damage to antibody - not sure if it applies to all in one's - also we use secondary fluorochromes as it gives more flexibilty (you always have the choice of an avidin/biotin amplification step).
dont forget fixed tissue will require epitope retrieval (triton x is good for frozen, boiling in citrate buffer for formalin fixed in parrafin)

dom

Ok, many thanks for your help! I will see what I can do!

0.2% triton x for ten minutes
do it before you block
not that many steps really

dom