PSD95 antibody and synaptic reagents: detecting a synapse, not a protein

Measuring synapses and transmitters

1. Score apposition, not puncta. Count pairs where a presynaptic and a postsynaptic marker are adjacent within the resolution limit. Counting one marker alone measures protein clusters, some of which are not synapses.
2. Fix for the compartment. Synaptic scaffolds and membrane receptors have different fixation optima, and a protocol that preserves one can destroy the other. Where both are needed, test the compromise on control tissue first.
3. Extract membrane targets deliberately. Channels and receptors are multipass membrane proteins that stay in the pellet with standard lysis and aggregate when boiled. Use a membrane protocol and follow the supplier's loading advice.
4. Separate transmitter identity from synapse number. Vesicular transporters assign the transmitter; scaffolds count contacts. Reporting a change in one as a change in the other is a frequent overreach.
5. Control the resolution claim. Apposition at the diffraction limit is suggestive rather than conclusive. Where the claim depends on it, use a super resolution method or an orthogonal measurement such as electrophysiology.

Resolution bounds what a PSD-95 antibody claim can be

At conventional light resolution, two puncta counted as apposed are within a distance that contains many non synaptic arrangements. That does not make the measurement useless, and it does mean the claim should be framed as putative contacts rather than synapses.

Where the number is central to a conclusion, add a method with better resolution or a functional measurement. Two independent lines make the claim durable.

Membrane targets are a different technique

Receptors, channels and transporters in this list share a problem with every other multipass membrane protein: poor solubilisation, aggregation on heating and epitopes that depend on conformation.

Treat them as a separate protocol within the same study, and validate each reagent on a positive control tissue before using it on the experimental material.

A vglut1 antibody and a vgat antibody separate the terminals

Excitatory and inhibitory terminals are told apart by their vesicular transporters, so a vglut1 antibody and a vgat antibody are bought as a pair and imaged together. Both give a punctate presynaptic pattern that should sit opposite a postsynaptic marker, and that apposition is the evidence the staining is synaptic rather than background. Fixation is the usual variable, since strong crosslinking buries these epitopes.

A nav1.7 antibody and a nav1.8 antibody in sensory neurons

The sodium channel family shares extensive sequence, and the subtype is the whole point in pain biology, so a nav1.7 antibody and a nav1.8 antibody both need cross reactivity data against the other subtypes. These are large multipass membrane proteins, which means gentle lysis, no boiling, and a membrane preparation rather than a whole cell lysate. A knockout tissue is the validation worth paying for.

A p75ntr antibody and an ngfr antibody are one receptor

The same neurotrophin receptor is catalogued under both names, so a p75ntr antibody and an ngfr antibody may be the same clone in two listings, and searching only one name hides half the market. The extracellular domain is cleaved, so an antibody against it and one against the cytoplasmic tail report different things after processing. The datasheet's epitope position is what tells the two apart.

Common questions

Is a punctum a synapse?

Not necessarily. Scaffold and vesicle proteins form clusters that are not all synaptic. Apposition of pre and postsynaptic markers is the standard evidence, and even that is limited by resolution.

Why can I not detect my receptor on a blot?

Because it stayed in the pellet or aggregated on heating. Multipass membrane proteins need a membrane extraction protocol and usually gentler denaturation than a standard sample.

Which marker, such as a VAChT antibody, assigns transmitter identity?

The vesicular transporter, because it determines what the terminal releases. Synthetic enzymes are informative and less definitive, since the enzyme can be present where the transporter is not.

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