How critical is glutaraldehyde step in postembedding? I think, secondary antibody can bind to primary antibody strong enough that glutaraldehyde post-fixation is not necessary. The reason why I am asking is that every time I use glutaraldehyde I am getting terrible EM ultrastructure.
Are you fixing your samples prior to embedding? I have never done a post-embedment fixation even though UA and LC are considered somewhat fixatives. Following secondary antibody, you should just be able to rinse and let the grid/sample dry, then image.
We always include postfixation of specimens with 1% Glu in PBS prior intensification of the 1.4nm FAB 2nd ABs and I guess that it is necessary, on the one hand to postfix the mild fixed tissue (4% PFA, 15% sPA, 0,05% Glu in 100 mM PB) and on the other hand to improve the AB binding. I don´t think that the postfixation could cause any bad structure probably the perfusion condition (fixativ & time of fixation) or the used buffers (we use 100mM PB and PBS). We obtain usually well preserved structure and play with our immunolabeling conditions (fixation duration), the label could be more intensiv if we decrease the structural integrity. It´s often a balance.
Which specimen are you fixing, most specimens are post fixed in Uranyl acetate or OT. i suggest you do a thorough search on post embeddg fixation.good luck.
Hi David,
You did not mention what type of protocol (or what type of EM – I suppose TEM) you are using. For most (99%?) of routine TEM embedded specimens post embedding post fixation is not needed and as any unneeded step in preparation may induce artifacts. Standard protocol: glut fixation, OsO4 post fixation, dehydration, embedding, cutting, staining works for most of the specimens.
We fix in different aldehyde combinations depending on the antiboidy and tissue. Glutaraldehyde will destroy some antiboidy epitopes we seek to identify (for example sheep prion protein) and for such immunoogld studies we will routinely use 4% paraformaldehyde alone. However, in our hands paraformaldehdye alone gives less good membrane fixation than does a mix of glutaraldehyde and paraformaldehyde. So when we look at murine disease, where the antiboides we use resist glutaraldehyde degradation, we will fix in 1% glut and 2% para , post fixed in OsO4, embedded in araldite.
I guess the answer to some extent is it depends on what strucutres you are investigating and what antiboides you have availalbe and to some extent you can only try different combinations till you find the one best suited to your purpose.
I understand that you are making an immunostaining directly on the grids (GABA likely), then you are using a silver enhancer kit or similar to enlarge the gold particles. In this case the GA postfixation is not compulsory but it will help to keep all the labelled antibody in place. In my experience I get more labelling after postfixation. The name of postfixation is misleading. It will do nothing on the tissue. If your ultrastructure was bad, likely it was due to bad perfusion or inclusion, check a grid under TEM before immunostaing to be sure (in this grid immunogold would not work afterwards). If bad ultrastructure is just because of low GA content, GA postfixation in the grid will not help, use more GA in the perfusion or look for an alternative (I'll need more information)
Hope it helps.
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