I am currently processing 400 um hippocampal slices immunohistochemically. I have not had too much trouble with my images and antibody binding. That said, I am aware that over fixation may interfere with antibody binding (i.e. due to excessive crosslinking). Currently, our lab fixes hippocampal slices in 4% paraformaldehyde for 1 hr, which is followed by 5 washes in PBS for 30 min at room temperature. Thereafter, we have been storing our tissue in 0.01% sodium azide in PBS. I would like some thoughts on our current protocol. What is the recommended fixation time for 400 um hippocampal slices in 4% paraformaldehyde? Also, how should these slices be stored after fixation and prior to immunohistochemical processing? Ultimately, I am interested in any thoughts on the fixation time of hippocampal slices and how it relates to the disruption of immunohistochemical processing.
Thanks in advance.
Hi Spencer,
RE fixation protocol I think you’re spot-on regarding the fixation time, I’d just recommend the following changes:
1. I think you should use PB instead of PBS (both for the 4% PFA and the washing solution). These names are often used interchangeably, but there is a difference between these solutions important to be aware of:
https://www.researchgate.net/post/Is_there_any_difference_between_PB_and_PBS
https://www.researchgate.net/post/Is_there_any_difference_between_4_paraformaldehyde_in_Phosphate_buffer_and_4_paraformaldehyde_in_PBS
2. Rather go for a higher number of shorter washes in PB (say 8 washes of 10 minutes) when removing the PFA.
RE storage: the 0.01% sodium azide in PB solution is fine for storage (in fridge at 4 degrees) if you intend to do the immunohistochemistry steps within a few days. For longer-term storage you may want to resort to freezing in some sucrose-based cryo-protection solution.
Hi Spencer,
RE fixation protocol I think you’re spot-on regarding the fixation time, I’d just recommend the following changes:
1. I think you should use PB instead of PBS (both for the 4% PFA and the washing solution). These names are often used interchangeably, but there is a difference between these solutions important to be aware of:
https://www.researchgate.net/post/Is_there_any_difference_between_PB_and_PBS
https://www.researchgate.net/post/Is_there_any_difference_between_4_paraformaldehyde_in_Phosphate_buffer_and_4_paraformaldehyde_in_PBS
2. Rather go for a higher number of shorter washes in PB (say 8 washes of 10 minutes) when removing the PFA.
RE storage: the 0.01% sodium azide in PB solution is fine for storage (in fridge at 4 degrees) if you intend to do the immunohistochemistry steps within a few days. For longer-term storage you may want to resort to freezing in some sucrose-based cryo-protection solution.
I agree with everything Matthijs said above!
Especially that more and shorter washes are better. This is even more true for the actual ihc/if.
I'll also add my 2 cents by saying that I think some permeabilization is crucial for thick slices. Even if you don't think you need it because your epitopes are all extracellular, it will help antibody penetration greatly.
Dear Spencer,
Do you use cryosections? I think the main reason for problems of poor antiabody binding is the poor penetration of the antibodies. 400 um is quite a distance and some way of permeabilization could be used. You could also use a complete different kind of fixation. In our hand fixation in Bouin´s fluid (1 h) followed be three changes in 70% ethanol and storing in 70% ethanol works sufficiently.
Regards, Daniela
We work with 400 um spinal cord slices (rat and mouse) and use a completely different approach: we re-section the slices using a cryostat.
Here is a summary of our method:
1) We fix the slices in 4% PFA/picric acid/PB at 4 C overnight.
2) Cryoprotect in 20% sucrose/PB.
3) Place slice on a coverslip. Cover with a drop of OCT. Place the coverslip with the slice on a metal block on top of dry ice. This freezes the slice inside the OCT instantly.
4) In the cryostat, precut a block of OCT on a chuck. Mark the top of the chuck to place it in exactly the same orientation.
5) Pull the frozen slice from the coverslip by pushing laterally with forceps. Put a small drop of OCT on the precut chuck. Press the slice (flat side down) on the drop of OCT to attach it to the chuck. Freeze inside the cryostat.
6) If you have done this correctly, the slice should be completely parallel to the cutting plane of the cryostat blade. This allow us to obtain 14-16 sections of 25 um from a slice of 400 um. You have enough sections to stain with different antibodies.
We do not have a problem with overfixation with a variety of antibodies, including neurokinin 1 receptor, mu-opioid receptor, Y1 receptor, substance P, enkephalins, etc.
We do all the washes with PBS in the 25 um sections, not the slice. Antibody penetration is the big problem here. Sometimes we have problems with antibody penetration even in the 25 um sections. A 30 min incubation with 50% ethanol in PBS can help with this.
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