Hi everyone, just ask some questions that I can't understand.
It is a common sense that fixation block antigen, especially for the tissue that should be fixed for a long time (such as thicked skin and lung).
In paraffin embedding, tissue are fixed well before dehydrate and embedding. Antigenicity are thought to be destroyed by fixatives in paraffin embedding tissue so one should perform antigen retrieval before primary antibody incubation.
In OCT embedding, tissue are directly put into OCT and frozen in liquid nitrogen, or pre-fixed and dehydrate using sucrose before embedding in OCT.
In general, paraffin section gives good morphology of tissue normally while frozen section gives good antigenicity and you can directly probe section with your 1st and fluorescent antibody without antigen retrieval if no long-time fixation before embedding.
However, recently one of my workmates tell me that they never use liquid nitrogen or dry ice to prepare frozen section, they just fix tissue overnight in 4% PFA and use 30% sucrose to dehydrate. Then, they use frozen section to do Immunofluorescence and get good results without antigen retrieval! Actually I was shocked!
If long time fixation block most of antigen, why do they do IF on frozen section without antigen retrieval? And the most striking thing is that they can detect GFP signals expressed by tissue immediately after section(they use transgenic mice that express proteins with conjugated GFP)! Why?
In addition, what is the meaning of cryosection if you fix tissue for a long time before embedding? I think it can neither give a good morphology or antigenicity.
Yes, this is especially true for formaldehyde, paraformaldehyde, glutaraldehyde fixation. That is why often the immunofluorescence protocols include an antigen retrieval (or antigen unmasking) step, for example using sodium citrate.
my workmate said even after 12hr pfa fixation，they can still detect fluorescence from gfp without the help of gfp antibody? is that possible，i am afraid that could be tissue autofluorescence?
Dear Jifan,
What kind of sample preparation are your colleagues using? Is this PFA/ paraffin embedding? Could you add more details?
Also, what are their negative controls?
Dear Gabriele
My colleagues use the following protocol:
1. fix tissue in 4% pfa o/n at rt.
2. 30% sucrose.
3. Oct embedding, store at -80.
4. cryosection, 10um.
5. observe fluorescence under microscopy.
I think maybe pfa fixation only have limited effect on Loss of antigenecity? It could be xylene, paraffin that really destroy antigenecity in paraffin embedding tissue ?
Hi Jifan,
I have a limited experience in OCT/cryosectioning. I used to prepare OCT-embedded sections to study retina development in Xenopus. In that case, i didn’t use antigen retrieval protocols and often i didn’t even need ImmunoFluorescence for overexpressed GFP, since in these type of preparations GFP retains its endogenous fluorescence.
On the other hand, for paraffin sections i used antigen retrieval and always antibody to stain Go. The dehydration steps included in paraffin sectioning kills GFP fluorescence.
* to stain GFP.
I also wanted to add: you said that you suspect your staining could actually be background/autofluorescence. In that case what kind of controls are you using?
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