[Histonet] Immunohistochemistry on brain: free floating versus cryostat mounted sections

[Lauren Tanabe]

Hi.  I'm new to histonet and have found it very helpful.  I was hoping 
to get some feedback on the following.  I'm currently trying to figure 
out the best way to do DAB staining on mouse brain from a variety of 
ages ranging from E 15 up throuh P 14.  If I try to do free floating 
sections on the younger animals, the sections fall apart.  At the same 
time, I just can't get my cryostat sections to look nice.  I've been 
perfusing with 4% PFA with a 3 hr post fix.  I'm thinking that this is 
probably not long enough.  I'm starting from scratch here so I'm trying 
to figure out:

1.  The best way to slice and stain embryonic through P14 sections

2.  The optimal thickness

3.  If using cryostat mounted sections: How to get my sections to stick 
to the slides better.  I invariably get little bits of brain that come 
up off the slide or maddening little bubbles underneath my section.  
(I'm using Fisher Superfrost/Plus slides)

4.  The best way to mount free floating sections.  The few times I have 
tried this, I get to the last step (with the help of mesh wells that 
fit into 12 well tissue culture plates) and when I pick it up with a 
brush I have a very difficult time transferring the section and not 
tearing it.
	--I also notice that I get little grid marks on my section from the 
mesh.  Not terrible, but I'd like to eliminate it if possible.
	--Also the best way to minimize background.  And when to do steps at 
room temperature versus at 4 degrees.

Sorry if some of these questions are naive.  I'm very new to the world 
of histology.

Thanks in advance,

Lauren

Columbia University, NY