[Histonet] question about immunofluorescence staining in
Andrea T. Hooper
anh2006 <@t> med.cornell.edu
Tue Mar 18 18:38:55 CDT 2008
For immunofluorescence, frozen sections are the easiest way to go
with the best results.
Acetone, methanol or a even a mix of acetone:ethanol are wonderful
ways to fix frozen sections of murine spleen for IF. A brief 2-4% PFA
fixation on fresh frozen splenic sections works also but the PFA will
increase the "autofluorescence". It will depend on the antibody which
fixation works best ...
Do you have access to a confocal or similar type system (one with
apotome or deconvolution software etc.)? If so, you can do 30-100 um
sections with beautiful results. For regular epifluorescence, we use
8-12 um sections with excellent results.
Let me know if you need further information/specifics,
>My boss has asked me about immunofluorescence staining in mouse
>spleen: How do you get rid of fluorescent background in mouse
>spleen? I suggested ammonia ethanol quenching which I know works in
>bone marrow. Is there a better method? Also, my boss claims that
>the macrophages have autofluorescence (formalin fixed paraffin
>embedded tissue). I always thought the rbcs would be the
>autofluorescent cells. Do the macrophages have autofluorescence?
>Finally, is it reasonable to do frozen sections of mouse spleen? I
>thought frozen sections fixed in ice cold acetone or
>acetone-methanol might solve the problem, but I was told that frozen
>sections would be much to thick to get any good immunofluorescent
>Thanks for any info and suggestions.
More information about the Histonet