Our lab has noticed that after several tissue preparations for organotypic hippocampal slice cultures, some of our tissue is developing what appears to be large holes between the CA1 and dentate gyrus. Has anyone using this model noticed this or have any suggestions on how we might address this issue?
We have checked CO2 levels in the incubator, and no changes have been made in our procedure or culturing media preparations.
Hi Caleb Bailey ,
There are many factors and questions that need to be addressed properly to troubleshoot this issue. For example:
How thick are your slices are and have you tested to see if slices with different thickness can have the same issue?
Have you checked the concentration and pH of the ingredients of your storage medium?
If you are preparing your slices for patch clamp, have you checked the temperature and possible fluctuation in osmolarity?
Does this also occur during patch (possibly due to the changes in pH, osmolarity, carbogen flow, temperature fluctuations...)?
Have you tried to check the sections under a microscope (preferably after a Nissl or H & E staining)?
Hi Bahman Sadeghi ,
Our slices are 200um thick, and we have been using this thickness in our protocol for 10+ years, so I'm not sure if that is the issue.
The concentration are controlled for and pH of the ingredients in our storage medium is adjusted to 7.2 at room temperature; this has also been the standard on our protocol for several years.
Our lab does not conduct patch clamp procedures. Usually our primary measure is immunohistochemistry which is imaged under our scope.
Under the scope is when we first noticed the distinct holes, but they are observable to the naked eye as well.
I've attached an image. The stain is for p-Tau Thr231 with TRITC secondary. The large holes should be evident here.
Thoughts?
Thanks,
Caleb
Thanks for the details Caleb Bailey ,
Since you have mentioned that only some of your slices have such tears or holes, any possible problem with your sectioning and microslicer or vibratome blade can be eliminated.
Please see if the intensity of the vibratome and vibration of the blade is consistant and suitable to avoid any holes or mostly tears.
I am not sure if the age of the animals can be an issue (if someone else have any thoughts on this...)
The thickness is also good and cannot be an issue. Though I would recommend testing slices with different thicknesses (100 and 400 um for instance) to have a more clear comparison. Though this could be mostly due to physical uneven pressure during slicing, please consider the following too:
You may search and examine different dissection buffers as well (e.g. 1 mM CaCl2, 5 mM MgCl2, 4 mM KCl, 26 mM NaHCO3, 8% sucrose, 0.5% phenol red, and filter-sterilized and bubbled with 95% O2/5% CO2 (carbogen). Possibly ice-cold buffers.
Also, different culture media, like this one that I found: MEM with Earle’s salts and l-glutamine (Gibco 11095) supplemented with 1 mM CaCl2, 2 mM MgSO4, 1 mg/L insulin, 1 mM NaHCO3, 20% heat-inactivated horse serum, 0.5 mM l-ascorbate, 30 mM HEPES, 2.3 g/L d-glucose, and pH 7.3.
After the brain removal (as quick as possible), if you use ACSF to embed the slices, then check the temp to be around 5 degrees Celsius.
Store and hold them at 35.0 ± 0.5°C in carbogenated ACSF prior your IHC to see if this helps.
Please let me know if the problem remains or solved.
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