I'm starting to do hippocampal slice cultures of P5-P7 postnatal mice. I am interested in the establishment of sprouting at the mossy fibers, by culturing the slices with pilocarpine. I have refined my culture protocol so that my slices seem to be OK by the time I have to check for the sprouting, about 5-9 DIV. I am labeling mossy fiber terminals with an antibody against Synaptoporin. My problem is that I think I'm having problems with the penetration of the antibody in the tissue, so that I can't label the mossy fibers properly (it seems that I have some kind of superficial labeling, but inside the slice I get a very weak signal), and even DAPI staining, to label nuclei, does not enter in the fixed slice. I have tried different concentrations of the permeabilization agent (Triton), from 0,2 to 1 % and permeabilization protocols (doing the immuno without pre-permeabilization, permeabilizing 6 hours before starting the immuno, permeabilizating overnight, etc), but I don't get advances. I know that it is not a problem of microscopy; I'm using a confocal and see green cells (I'm using a strain that expresses GFP in pyramidal cells of the hippocampus) in the deepest part of the slice. Can anyone help me with that?
Thanks in advance
Sergio,
See the linked reference. I have not attempted this yet, but it may address the problems you have been having:
http://www.microscopy-analysis.com/sites/default/files/magazine_pdfs/mag%20%202011_January_Yoon_0.pdf
Alternatively, though technically difficult, some references describe embedding and slicing the hippocampal slice to get antibody penetration.
D
Thank you David:
I had seen this pdf before, and I tried the overnight incubation with Triton 1% before, but it didn't work...
I have thought about slicing mi slices, but I wanted to ask here for other posibilities before.
Any other idea?
Thanks
Hi Sergio,
I might be a bit late since May, but anyway...
How long are the incubation times? With thick sections (>200um) you should increase the incubation and washing times to allow penetration and exchange of molecules through the tissue. In the past, I was using these times for staining ~250um-slices: 6h for the permeabilization and washing steps, 3-4 days for the antibody incubation, 4-5h for DAPI... I can send you some more details if you need. I am attaching also a helpful reference.
Anyhow, it might be worth it embedding and sectioning your slices in a vibratome, especially if you want to go for very high magnification at the confocal microscope, because the short working distance of the objectives might make it impossible to reach the center/bottom of your slice.
Hope it helped!
Hi Sara:
Thank you very much for your help. I've tried different permeabilization protocols, from 4 hours to overnight, and 2 days for the primary antibody (for the secondary only 2 hours, as I always do in my immunos). I think that the problem I have is that there is a layer of glia covering the slice and preventing the antibody to enter. I have also tried to embed and section my slices, but it didn't work. I saw your reference, thank you for sending. I've seen that they use MeOH in PBS for 5 minutes after fixation (I use to fix the slices for 2 hours, I've seen they do only for 5 minutes), do you know what MeOH does? Do you use it?
Thank you again for your help
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