Our tissue sections are firstly used for microelectrode assay (MEA) recordings and then for immunofluorescence/immunohistochemistry and wide field microscopy. We've already tried 300μm because that is efficient for the MEAs, but we couldn't see anything clearly under the microscope. My lab tried in the past 100μm thickness, but the slices aren't good enough for MEAs.
For immunostaining 20µm thickness works well with most antigens, including pre- post synaptic proteins, axon initial segment and cell-type markers.
I tried it as post hoc following regular electrophysiology experiments (250-300 µm thickness), I incubated the slices in fixative for a brief amount of time then cryoprtected sections in 30% sucrose overnight. Next day, I sectioned them in thinner slices (20µm). If you have further questions, please do not hesitate.
For immunohistochemistry (did it on lung tissue) I use 5-10 μm, but this is probably too thin for MEA
Dear Konstantinos,
One of the issues may be with MEA recordings is damage to the tissue (surface). I regularly use brain slices and whole-mount retina (~300 micron or more thickness). I record and fill multiple cells using single or double microelectrodes in each preparation and I subsequently fix and do immunohistochemistry with upto 4 labels with no major problems. The main limitation is the high magnification (63x and 100x) confocal imaging in tissue below 30-40 micron depth, no problem with the top 25 micron. Therefore if you like you can further make sections from your slices at 20 to 50 micron intervals once they are fixed to optimize confocal imaging at high magnification. However, keep in mind that if you want to overlay images from individual sections, it is time consuming and difficult job. See my following publications at RG for details of my protocols.
Best wishes,
Refik
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You can do it in two ways:
1) Do some thin sections for Immunocytochemistry only. 4 to 7 micron sections would do. Then try to correlate with your thick sections.
2) If you have to use thick sections then use other methods of imaging such as two-photon confocal or deconvolution with wide-field fluorescence. Both are good for thick sections.
Regards
Normally I used 10-20 µm sections for different Imunnohistochemical analysis in plant tisssues with good results. I think, that your mentioned 300 µm are too thick for your microscopical approach. Tahani k Alshammaris suggestion is a good solution for the realization of MEA and Immunohistochemistry on the same sample.
I also follow similar method like what Tafani described when I run both acute slice cultures (to treat them with different drugs first and then stain them) , fix them with 4%paraformaldehyde-PBS, make a block with OCT compound, section with cryotome to make them free-floating sections to label them with different antibodies and finally view them with microscope. Hope it helps.
I can recommand Tahani's method. I worked with this method for a long time. We detect biocytine labelled cells in combination with IHC or Immunfluorecence and it works pritty well. We use 25 - 30 µm thick frozen sections and use free floating staining method. Try it and I promise you you will get good results. Best wishes Ute
Hi Konstantinos,
To my mind, you should follow the whole mount staining protocol.
good luck
Hi Konstantinos,
To my mind, you should follow the whole mount staining protocol.
good luck
For more information about conducting electrophysiological experiments and Immunolabeling from the same brain tissue, take a look at this method paper published in Frontiers in Cellular Neuroscience.
Tahani
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