Hi Tara, I came across your question when I was querying this myself. On the 2 occasions I have made the Walton's Lead I have found that the reason it has not worked or not remained in solution was due to the stock solution. I had prepared a stock solution pH 3.8, however I found it preferable and for it to work when preparing it all fresh. I add 0.06g of lead nitrate to 10ml of the stock, it remains clear and you can see it dissolving but it should not go cloudy (which I was finding). This is all done at room temperature, and then it is pH'd with 1MKOH dropwise to reach 5.5. It should be swirled gently between each drop, and not have a magnetic stirrer and this may cause precipitation. Swirl until clear and until desired pH is reached, then it goes into a pre-warmed 60 degree oven for 30 minutes. It is then syringe filtered through 0.2µm and used to incubate the samples which go back into the 60 degree oven, for the protocol I use, it is incubated for another 30 minutes.

Thank you so much Rhea. It sounds as though your protocol is very similar, only I add KOH to the buffer the night before (using aspartic acid), which will bring the pH from somewhere around 3.5 up to 4.1 or so. When making the lead staining solution, it does typically go into solution immediately. Then we warm it for 30 minutes, add it to the tissue, and leave in the oven at 60 degrees for another 30 min. It has been working very nicely for contrast in EM work, so I am not going to change anything.

No problem! Sorry it's a very late response to your question, but thank you kindly for responding so soon. That is interesting, I may attempt this if it works for you (adding the KOH to the buffer the night before). Yes I am also using this for contrast staining for Volume EM work which I am still quite new to but enjoying it.