[Histonet] Some questions about dehydration and clearing

[Allan Wang]

Hello,

I've been doing some histology work but without formal training. Just some
questions about protocol:

1) Is it okay to dehydrate directly into 100% alcohol or does that cause
tissue shrinkage? Is starting at 95% good enough? I'm trying to reduce the
number of stations needed.

2) I've once been told that after dehydration, I should wait for all the
alcohol to dry off before clearing with xylene. Does that make sense, since
I couldn't find this step in any protocols I've seen. Or would this mess up
morphology?

Sometimes I see some inconsistent appearance of cells after IHC staining,
not sure if it's due to issues with this dehydration step/clearing or if
it's the change in antibody dilution that's causing it.
I have a comparison image here, where I think something went wrong with
Slide 2. Both IHC was performed with an autostainer.
http://i.imgur.com/y0q4hb7.jpg

Thanks,
Allan Wang