Yes, formalin-fixed brain tissue (perfused), wax-embedded. Pre-treated microtome sections in Boric acid and use Sy38 from millipore. Used this successfully in Cunningham et al., 2003 European Journal of Neuroscience and elsewhere.
Hello..you can see my paper, I have done the same, this may help you. If you have any problem during IHC, you can ask..
http://www.ncbi.nlm.nih.gov/pubmed/?term=Caffeine+and+modafinil+given+during+48h+sleep+deprivation+modulate+object+recognition+memory+and+synaptic+proteins+in+the+hippocampus+of+the+rat.
Your request is an "eye roller" everyone will point you to their paper. There are as many protocols out there as there are papers on stained synapses. Find one that suits your needs and here are some guidelines to help you figure that out:
Antibodies Inc. or SySy have good synaptic antibodies...typically. Their Synaptophysin antibodies are fine (Antibodies Inc is less expensive) and I know the Millipore antibody works great too.
If you are working with mouse tissue find an antibody raised in Rabbit and you may not have to do transcardial perfusion but I would recommend the perfusion anyway (use heparin in your washout before fixation!).
After sectioning and before staining, fix the sections for 5-10 minutes in 4% PFA.
Antigen retrieval is unnecessary for good synaptophysin staining just don't make your sections too thick (15-50um). and use 1% triton-X100 or tween-20 in your blocking buffer.
If you put 0.025% sodium azide into your blocking buffer you can primary stain at room temp overnight. I get better staining this way but you can do it at 4 degrees too.
Do not, do not, do not secondary stain for longer than 1 hour. Bad things happen...bad things. i.e. lots of background fluorescence and a wasted day of work.
Tissue clearing with Xylenes and rehydration can be a little tricky to get right and if you can learn it great. Just use a good mounting medium and don't cheap out.
Image your tissue promptly for the best data.
cheers and good luck!
I would like to analyse the effect of LPS on synaptophysin in mouse hippocampus and cortex. Could anyone recommend the sections that i should look into, thanks
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