Re: [Histonet] (kinda) silly questions about IHC

From:Helen Fedor

If you wanted to cut free floating sections and mount them on slides
and dry them over night. you can do that.
I cut 30 micron sections this way and did LFB/PAS. I may have gotten
down as far as 25 microns. I don't think I ever went any thinner than
that on free floating sections.
For Immuno the problem is you will not get the staining from both sides
of the tissue.
It can only penetrate through the surface and not from the side that is
touching the glass. Dab is a large molecule and will not penetrate very
far. That is why the Floating method is used. Double the amount of
Try to slow down your agitation if you can, that may help if you want
to give the free floating another try.
I have cut many frozen cryoprotected sections onto slide. start with
cold slides.
put the section onto the slide keeping it in the cryostat. The section
is melted onto the slide very slowly using the heat from a finger on the
back of the slide. Start at one end of the section. as it melts slowly
slide your finger toward the frozen part(this is all on the back of the
slide). While doing this use a brush on the section. and slowly brush
the section down trying to keep it flat. this is all done inside the
cryostat so every thing is cold. Only the heat from your finger melts
the tissue
It is a lot of hand and eye coordination to get this to work. But it
can be done.
This is done on 10 micron sections. good luck
>>> "SMITH,REBEKAH FELICIA"  10/29/04 11:15AM >>>
I don't know if this will help, since all I know about is paraffin 
sections, but I mount my tissue on slides from a water bath (which 
will keep bubbles from popping up in between the slide and the 
tissue as long as the water bath isn't boiling or otherwise bubbly 
itself) and leave them to dry overnight on a slide warmer. It's 
never hurt my paraffin sections to let them dry like that, but I 
don't know how that compares to frozen sections.
                                 Rebekah Smith
On Fri Oct 29 09:52:08 EDT 2004, Anna Elisse Beaudin 

> Hello,
>    I have a question about IHC.  I am trying to do multiple 
> labeling on
> sagittal brain sections that were fixed by perfusion.  I cut 
> these
> sections on a cryostat, and I am having a lot of trouble deciding
> whether to do IHC on mounted sections vs. free-floating.  When I 
> try to
> mount fixed sections directly while sectioning, I get air bubbles
> caught between the section and the slide.. the sections just 
> don't seem
> to want to lie smoothly on the slide.  Alternatively, when I 
> stain
> free-floating sections, these long sagittal sections curl up and 
> as
> such I get extremely uneven (and ugly) staining.  My actual 
> (silly)
> question is whether it is possible to collect sections into 
> solution
> (free-floating), mount them, let them dry overnight, and then do 
> IHC on
> them.  I'm torn because I need to let the section dry a little on 
> the
> slide so that they stick, but at the same time I think it might 
> be bad
> for them to dry out?  As you can see, I am very confused, and 
> would
> really appreciate anyone's advice.  Thanks in advance for your 
> help!
> Best,
> Anna Beaudin
> Division of Nutritional Sciences
> Cornell University
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