Hello Irena,

you have to fixat the tissue very properly in 4%PFA or Formalin. This is absolute essential for silver stains, otherwise it does not work.

You have to be very carefully with OsO4. OsO4 is a hazardous substance. You have to work under the hood. Waer gloves and a lab-coat. Put the OsO4-solution into a jarr with a twist-off-Lock before you put it into a fridge for storage, otherwise your fridge is getting black. Even the microtiter plates or petri dish, which you use for the reaction seal carefully with parafilm and put it in a paper box during the reaction.

The Ag No3-solution is not so dangerous but it makes black spots which can not remove with water and soap, so wear gloves and a lab-coat even when you are working with this solution.

Ute Neubacher thank you very much for the thorough response, I appreciate all the suggestions you gave!

I just have one last doubt about the fixation: I work with mice and usually after harvesting I post-fixate the brains 15% sucrose in PFA (4%) overnight, next 15% sucrose in PBS and then to 30% sucrose in PBS, each for one day. Would that be enough for the staining to work or should I start considering perfusion?

Thank you once again!

Bests,

Irena

Hello Irena, your fixation protocol is not enough for the silver stain. I would recommand either perfusion or immersion fixation in 4%PFA for appr. 1 week.

Post fixation after perfusion 2 days min. Kryoprptection with sucrose is not necessary because you use the vibratome for sectoning. If you don't do anything else with the tissue e.g. like immunohisrochemistry you can store the brains in the fixative until your start with the sectioning. 2 days before put the brain in PBS . You can also use the fixaton protocol which is mentioned in the Lanz paper.

Ute Neubacher, thank you very much once again for all the insight!

I will gladly follow your suggestions!