Hi Emanuela, 

First, on which type of preparation do you plan to use? In our lab, we worked preferentially on whole mount tissue preparation.

Whole mount will be fine. I am new in this field and I always did SNC staining. I saw some protocols where the tissues are first perfused and others instead fix the tissues postmortem. What do you recommend? My aim is to do a co-staining of the dopamine neurons with alpha-synuclein in a model of Parkinson's Disease both in the myenteric and submucous plexus.

Hi, 

For SNC, I think perfused tissue is best but for gut you can fix it post-mortem. When you sacrifice your animal, you remove the intestine and place it in a cold HBSS or Krebs solution. In a glass petri dish containing Sylgard coating, you fix your tissue with minuties and detubulized it along the mesenteric side (you can also do it without attach your tissu if you're good with microdissection tools). Once your tissu is detubulized, you need to strech it a lot with the mucosa in front of you before fix it with 4% paraformladehyde. A good fixation is obtained after 3h at room temperature or 24h at 4°C. For the microdissection part, it's more difficult to tell you how to do it, you need to get a formation by an expert in this field. It's easier in rodent than in large animal and human but still not easy to do. Briefly, you need to remove the mucosa part without scratching the submucosa to remove it safely and make correct staining of submucosal plexus. Once you will remove the submucosa, you'll have access to the smooth muscle part of the tissue. The myenteric plexus is between the two layer of muscle (circular muscle and longitudinal muscle). Depending of the thickness of your muscle layer, you can do immunostaining without removing circular muscle layer (myenteric plexus is mainly attached to the longitudinal muscle layer). For better immunostaining (muscle cells can produce a lot of background), you can safely remove circular muscle fiber.

You need the correct microdissection tools and work under dissecting microscope. And feel patient to make a good dissection!!! Good luck

I think you can harvest the tissue you need, fixed with 3.7% PFA and used cryo microtome to cut and mount on the slides, this will be easier for you to do the staining and also do use a charge glass slides, and bake in the 37 degree incubator for at least half hour to stable the attachment of tissue on the slides

Hi Emanuela

colleagues above have answered most of your questions and agree with their recomendation. Perfuse fixation usually limits the strechebility of the gut tissue and learning to strech the tissue optimally witout breaking the internal and external layers of the gut is the first most important step for producing a good wholemount preparation. The next hurdle is separating the muscle coats to expose the enteric ganglia. Guinea pigs are the best samples to work on muscle coat separation as they contain natural clevage points between the muscle coats we need to separate. This is however not true to the other animals including human gut. Therefore one has to carefuly remove the muscle fibres from the thicker muscle side as described by Julien.