[Histonet] penetration problem with floating brain sections

[Bass, Caroline]

Hello All, 

I’m doing some IHC with DAB on formaldehyde fixed rat brain floating sections, either 35 of 50 um thick. Usually I get fine staining of TH for example, but now I’m going after a neuron that's a bit sparse, and I noticed I have a couple of darkly stained cells. But I can see dozens of “ghost cells”, these appear to be very lightly stained in the right place. My guess is that the antibody isn’t penetrating fully, and so cells on the face of the sections are staining darkly, and some on the interior are just barely been stained. Any suggestions on improving penetrance? I usually have primary on overnight, shaking in the cold room, and secondary at RT for 2 hours. I was thinking of extending the primary to room temperature and maybe upping the triton-X concentration. Any other suggestions?

Secondly, I noticed were getting some cracks in the tissue. We usually mount the DAB stained sections on some sort of tissue bond slide, and then let it dry overnight before dilipidating and coverslipping with permeant. My best guess is that sometime during the drying or coverslipping the tissue pulls away slight, resulting in these smallish cracks.

Anyway, any advice on handling these two issues would be greatly appreciated.

Thanks,

Caroline Bass
University at Buffalo