Experimental Design Question for Immuno with Free-floating sections

[wsnow]

Hi all,

I am trying to do immunostaining with 6-week old fixed brain tissue and have tried frozen sections from a cryostat and free-floating sections from a microtome.  My question has to do with general research design. With free-floating sectioning, I would have to sacrifice as many animals per day as I can cut on the microtome.  Is it generally assumed that it is better to cut all your tissue over a few days (if needed), store the sections as taken in the fridge in PB and azide, and do immunostaining on the entire batch of sections after sectioning is complete vs. sectioning, then staining the day's worth of sections, and repeating the process until I have enough animals for the experiment?  I hope this makes sense. 

Thanks so much for your thoughts!
Wanda

[nzkat]

Hi - you can section your tissue on different days and store it either in the fridge with azide, or at -20C in a cryoprotectant solution (30% sucrose, 30% ethelyne glycol in 0.1M PB). If you want to compare the results between brains, it is important to do the immuno from all the brains at the same time. Otherwise variation in the immunos done on different days might look like a result when actually it isn't. If you have so many sections that it is impossible to do them all at once, make sure you include one lot of sections from the same brain in each immuno you do, to check the consistency of your results.
Kat

[wsnow]

Thanks so much.  I agree but just wanted to get a sense of what the standard was.  Much appreciated!

Wanda