Hi Aloysius

I don't see any logical method to quantify from fixed cells on the neurotransmission level. If they are fixed it means you have mainly immunolabeling as your main option of detecting something in those cells. Immunolabeling will mainly work on enzymes/proteins taking part in the biological production of the neurotransmitters you are interested in, but not single-molecules of neurotransmitters. Labeling those proteins can give you an assessment about the neurotransmission process, but will not be direct measure of neurotransmitters quantity. 

So you can use antibodies such as GAD67 to learn about the production of GABAergic neurotransmitters for example, and VGluT1 for glutamatergic.

Also, pay attention that if you are going to stain for the suggested markers, it is important to know the cells source in order to define the exact proper marker, as it can be changed.

Best

Boaz

 Thank you Boaz! that helps. yes fixing kind of takes off the table most of the options. Although GAD67 and Vglut1 as indirect methods, I think something is better than nothing. I will try to redo a trial without fixing my cells to see it can be done.

Cheers

Aloysius

Something like Western blot, you have to find an internal control if you want to quantify your immunostaining. For most of the time, you may see differences in fixation between animals or wells of cultured cells. The perfusion needle direction, blood vessel development and cell growth condition may vary between cases, which will give you lots of differences. That is why you cannot just compare the intensity or brightness of target cells or synapses in you stained samples. If you chose a marker such as NeuN, it may be more scientific if take a ration of your targets to NeuN.

For GABA staining, you should fix your cells with 4% PFA plus 0.125%-0.25% GA because GABA antibodies are usually made from GA-conjugated. So, I think you can do double staining with pairs of GABA+NeuN and VGLUT1+NeuN. You can also use DAPI or Hoechst to counterstain your samples.

For your reference, you can read the following paper.

Brain Res. 2012 Sep 20;1474:40-9.

Good luck. 

Hello:

Perhaps you could consider taking a small fluid sample to quantify these neurotransmitters in a kind of push-pull procedure for not to disrupt your fixed cell if they are alive (cell culture), I have a method to measure at least glutamate with high temporal resolution and in real time, please see this reference: Talanta. 2015 Nov 1;144:1231-8. doi: 10.1016/j.talanta.2015.08.013. Epub 2015 Aug 6.