So I have been trying to find good antibodies for detection of excitatory and inhibitory post synaptic elements in mouse brain tissue (perfused, 100um slices)
We are studying a different protein alongside in which the signal is ablated by antigen retrieval so we are trying our best to find antibodies that could work to label post synaptic densities on neurons.
Does anybody have success with this? recommendations of antibodies or alterations to my protocol?
Anything could help, please!
example protocol
sacrifice animal, perfuse with 20mL PBS, 10mL 4% PFA
fix tissue overnight in 4% PFA, section tissue
immediately add to perm/block solution for one hour (5% triton)
overnight primary antibody incubation (in .001% triton)
3-10 minute wash steps
4 hour secondary incubation (.001% triton)
3-10 minute wash steps
I can recommend a number; just three examples
inhibitory synapses: gephyrin (works really well)
excitatory synapses: homer, shank2
I can send you the company references and the full list by email.
Oh great! We actually have Gephyrin and Homer1, after looking through a few others suggestions on different threads, seems like I need to do a quick fix 30 minute PFA treatment instead of overnight.
do you have any technical recommendations for protocol? Would you agree with this advise?
thank you so much for the response!
-Jordan
You may want to check this nice piece by Ipolito and Eroglu 2010 (attached below). They recommend against perfusing with a fixative, which sort of goes along the lines of what you've already come across by yourself.
Gephyrin and Homer-1 are great choices for inhibitory and excitatory synapses respectively. Synaptic systems offers a solid ariet in species with very good performance and validated in KO.
Best of luck
Juan
Article Quantifying Synapses: an Immunocytochemistry-based Assay to ...
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