Re: [Histonet] question about immunofluorescence staining in mouse spleen

From:"Andrea T. Hooper"

Hi Sharon,

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,

>Dear experts,
>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 
>pictures on.
>Thanks for any info and suggestions.
>Unknowledgeably yours,


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