[Histonet] Dulbecco's PBS

[garciaa <@t> mskcc.org]

Hi, everyone.  I have a few questions I'm hoping you'll be able to help me out with.  I have 10+ years doing immunohistochemistry on PFA perfused, frozen brain sections, but a few things I'd like clarified.  

1)  Does it matter whether one uses PBS with or without Mg and Ca for immunos?  I have always done it with PBS without Mg and Ca, and have never heard of anyone doing it with Mg and Ca, but have recently encountered a scenario in which this was the only PBS available.  Wondering whether this will make a difference in my staining.

2)  Can anyone explain the need for dehydrating and defatting slides (after using such substrates as DAB for color development) before coverslipping?  Aside from the obvious that the sections look pretty crappy without these steps, can anyone explain the science behind this?  

3)  Because my tissue is perfused, I typically store the brains in 30% sucrose (with a little bit of sodium azide) in 4 degrees, until I am ready to cut the blocks.  At this point, brains are usually frozen in the cryostat just before cutting 40um thick sections.  I have obtained wonderful staining and morphology with this technique, however I have been recently advised to store the brains embedded in OCT (or similar) in a -20 degree freezer, rather than in 4 degrees.  I am skeptical and concerned that this will damage the morphology and produce that "swiss cheese" freezer artifact (you know, where there are all these holes in the tissue?)  Can anyone provide any advice, insight as to the benefits and drawbacks to either/both of these methods?

Thanks everyone!  Any advice, insight, comments are greatly appreciated.

D. Garcia

 
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