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

Dear All,

Thanks to everyone who responded to my question about 
immunofluorescence staining in mouse spleen and elimination of 
background.  The specific protocols and the info about macrophages 
taking up fluorescent dye nonspecifically will be very helpful.  I 
relayed the info (the question was for someone else's project) and I 
think the suggestions will be tried.  Also, I plan to start trying 
some stuff on mouse spleen myself - I've always avoided it and used 
bone marrow but your suggestions make it seem feasible.



>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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