C57BL/6 mice seems to be easily stressed during the daily handling and possible transfering during behavior test. How do you do to decrease the amount of IEG induction in rodent brain which are not related to the behavior test of interest?
For example, I want to know how formalin induced pain activate some subcortical regions which are involved in pain pathways. So I applied formalin to mouse hindpaw in the behavior test room, and then transfer them to the perfusion room where you have a good hood for ventilation. Here the problem comes. I found a substantial variation in expression of pERK (have not tested c-fos yet) in the same brain region, within control group(naive group &saline group) or same formalin group 90min after injection.
I have not stained for pERK before, but I have stained for c-fos and Arc. Both are IEGs that are induced by neuronal activation (which can take >30 min), unlike pERK, which just needs to be phosphorylated (which can take seconds - minutes). If you move your mice to the perfusion room and perfuse within 30 mins, you should see not see any of the activation caused by the move, when staining for c-Fos and Arc. I tested this a while ago, by staining for Arc protein and Arc mRNA simultaneously (perfused 60 mins after behavior assay). I saw much more mRNA than protein. The mRNA that was not colocalized with protein was a result of the transfer to the perfusion room and anesthesia.
I would also expect phosphorylation events to be easily reversible, which may lead to your high variability.
Another note: the c-fos antibody previously offered by calbiochem, which was amazingly strong and wonderful, and could be used at 1:20,000, is no longer offered. They have a new lot number and the antibody barely works. In addition, I'm not sure if there are any great Arc antibodies because I used to use a homemade one that is in very short supply now. See other thread.
https://www.researchgate.net/post/What_are_the_best_antibodies_commercially_available_for_C-fos_or_Arc_immunohistochemistry
I have not stained for pERK before, but I have stained for c-fos and Arc. Both are IEGs that are induced by neuronal activation (which can take >30 min), unlike pERK, which just needs to be phosphorylated (which can take seconds - minutes). If you move your mice to the perfusion room and perfuse within 30 mins, you should see not see any of the activation caused by the move, when staining for c-Fos and Arc. I tested this a while ago, by staining for Arc protein and Arc mRNA simultaneously (perfused 60 mins after behavior assay). I saw much more mRNA than protein. The mRNA that was not colocalized with protein was a result of the transfer to the perfusion room and anesthesia.
I would also expect phosphorylation events to be easily reversible, which may lead to your high variability.
Another note: the c-fos antibody previously offered by calbiochem, which was amazingly strong and wonderful, and could be used at 1:20,000, is no longer offered. They have a new lot number and the antibody barely works. In addition, I'm not sure if there are any great Arc antibodies because I used to use a homemade one that is in very short supply now. See other thread.
https://www.researchgate.net/post/What_are_the_best_antibodies_commercially_available_for_C-fos_or_Arc_immunohistochemistry
In addition to the above comment, which makes excellent suggestions, I would, If possible, anesthetize the animals in the behavior test room, prior to moving them into the perfusion room. That would minimize even further any disruptions that could activate the IEGs.
C57BL/6 mice are very sensitive, is it possible to change mouse strain? E.g. Swiss albino were more robust.
I agree with Manuel Alcaraz-Iborra, saline injection itself can upregulate fos or p-erk hence, we dont use a pinch test to see if the anesthetic has kicked in before proceeding with perfusion. you can have a another control group with no injection at all and see if the number of erk cells change. good luck
Please keep in mind that most anesthetics strongly regulate pERK/MAPK signaling (among other pathways). For references, see:
Article Brief Isoflurane Anesthesia Produces Prominent Phosphoproteo...
https://www.frontiersin.org/articles/10.3389/fncel.2019.00219/full
Based on my experience, these phosphorylation changes due to anesthetics are global, and can thus have significant confounding effects on whatever you are trying to study.
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