I am trying to figure out the optic time for biocytin filling with whole cell patch pipette. One protocol was saying about 5min, longer time will lower the success rate (Nat Protoc. 2006 ; 1(4): 1977–1986. doi:10.1038/nprot.2006.261). The other was saying at least 10min at 34 degree, longer time needed for room temperature (Article Immunostaining of Biocytin-filled and Processed Sections for...
). My concern is more about the success rate, not the details of the processes of the neuron. Please share your experience. Your inputs will be greatly appreciated.
Dear Jianli,
In my humble experience it was enough 10 to 15 min of wc mode (34 degrees, 2-3 layer neocortical pyramidal neurons) duration to see afterwards good biocytin development (i.e. processes were filled).
I thought more tricky part was to fill the cell with biocytin after cell attached mode (RT, striatal holinergic interneurons, CA1 pyramidal neurons). Since pipette solution was similar to bath solution continuous wc for biocytin filling could irreversibly affect cell membrane (it was just my speculation). But after 3-5 min of wc most of cells was filled well enough. For most cells processes were not fully filled.
Hence, another tip: since you don't need to have all the processes filled you can just observe holding current stabilization. It suggests that the cell body interior and pipette solution are mainly mixed and thus the cell is filled with biocytin . For processes filling one should wait for probably 10 to 15 min (as in my experience and in the article you mentioned)
Hope this helps. Good luck!
Dear Tigran,
Thank you very much for your very helpful inputs!
The "Nature Protocol" paper I mentioned above is saying that with longer time recording, it seems the membrane getting leakier, biocytin will leak out. That's why they suggested ~5min filling will have better success rate. I am wondering if this is true.
Thanks again!
Jianli
Typically the shorter time the better due to leakage from the cell/electrode. Optimal results will likely be determined by your subsequent protocols (staining and imaging). I have gone as long as 40 minutes after break in with success as we were interested in some getting functional data from the cells. Also, the less positive pressure you apply when inserting the electrode into the bath/slice, the less you are spraying biotin onto surrounding tissue which will help to reduced background.
Hi Daniel,
Thank you very much for sharing your opinion!
I am trying to record from the interneurons in spinal cord, then do IHC staining for cell type classification. The higher biocytin staining success rate the better to use the whole cell recording data. What's your suggestion for the recording time?
Thanks for your help!
Jianli
Dear Jianli,
In whole-cell recording mode, I recommend that you do your electrophysiological recordings first. Biocytin will passively move from your pipette to the cell soma and processes. To make sure that the soma and the processes are dye-filled well, I would actively pulse (depolarizing pulses) at the end of recording. These pulses can typically be around 500pA and 0.5s duration at 1Hz for 3-4 minutes. If you can pass 1nA then you can dye fill your cell in about 1.5 to 2 minutes. In other words amount of current you can apply (pass) determines the duration of dye-filling. However, higher current or voltage pulses can also damage your cell and reduce the recovery rate. There are also other factors critical for higher recovery rates of dye-filled cells. For detaiils and discussion, please see my following publications.
Best wishes,
Refik
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Hi Jianli,
Great question, and I agree with all the points above!
I'd just like to add that in our experience, successfully forming an outside-out patch is a good indicator that you'll retrieve the marked cell. So we are always super careful and slow as we pull away the electrode from the cell.
Especially if you do not need process labeling, I think you should not worry about pulsing biocytin into the cell, as this may give you more problems and will really only help with more distal staining...
Dear Refik and Alberto,
Thank you so much for sharing your experience and suggestions! They are very helpful! I really appreciate.
Jianli
Passively filling during whole-cell recordings I have two different experiences with two distinct cell-types. With pyramidal neurons 15 minutes was ideal and smaller neurons in the vlPAG (~10pF) are sufficiently filled after 5 minutes. In my experience you need to have it long enough to diffuse to all processes while still maintaining proper health to retract pipette and avoid beading or other issues. I also place slices directly into 4% PFA after retracting the pipette to fix slices ASAP.
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