[Histonet] need help staining 120um human whole brain sections!

[Maria Mejia]

Good morning Rene,

I have worked out special staining IHC protocols to work on celloidin cut sections from 60um to 600um thick.  These are 
free-floating human whole brainstem & whole brain sections. The IHC is amazing using single, double & triple. We save on
antibodies, ABC, polymer, TSA using the parafilm method. 

For example:
1)  I use a sheet of parafilm slightly larger than the section to be stained & it’s placed on flat surface (glass slide or dish
or whatever). 
2) Using a pipette, small drops of working diluted antibody are placed on the parafilm surface to match the size of the tissue 
section (from 500ul to 1ml). 
3) Then the tissue section is free-floated from PBST/2% Triton onto another cut sheet of parafilm, where carefully the section
is floating onto the middle surface of the antibody drops by using a brush. As the section lays flat, the drops spread throughout
the tissue surface - evenly covering the bottom of the tissue section.
4) This step is repeated for the top surface & the section is gently sealed by placing another parafilm sheet on the top surface
of the section to spread the antibody & seal the section for incubation overnight.

This method works, although time consuming! The problem is for the washes, quenching & blocking - we need a semi automatic
IHC system to take of these steps.  To bad there isn’t a robotic arm we could have built & programed to do the above steps. We
need an inventor type person to build what I can imagine.

best
Maria


> On Mar 20, 2017, at 6:51 AM, Rene J Buesa <rjbuesa at yahoo.com> wrote:
> 
> You have a special project → special tasks so your approach has to be equally special.
> Large brain sections are usually stained while floating but for IH with different and successive steps requiring very expensive reagents, floating sections is not well suited.
> You should affix the sections to large slides, and I imagine will not be brain whole sections but limited to some areas.
> In that case for IHC you can stain several sections in a humid chamber, manually. I do not imagine an automatic system for this task.
> It will be a costly and slow process indeed.
> René
> 
> 
> On Sunday, March 19, 2017 8:27 PM, Maria Mejia via Histonet <histonet at lists.utsouthwestern.edu> wrote:
> 
> 
> 
> Or lab is currently processing a human whole brain.  In about a month or two, the whole brain, which will be encased 
> in celloidin & serial sections will be cut at 120um each.  Now, we’ve bought an old Tetrander cast iron microtome. If 
> you haven’t seen one of these microtomes, I can tell you it’s BIG!
> 
> Now, we’ll have to stain quite a number of these sections for IHC.  In fact too many to handle manually. If possible, need
> to find a way to at least stain the majority of these sections in a semi-automatic system e.g. washes, quenching & blocking. 
> 
> Does any one think it’s possible to convert a LABGO processor (made in India & can hold 2 liter glass beakers) or a 
> Sakura-Tissue-Tek 4640 (also holds glass beakers - both are old style circular processors using glass beakers for staining 
> these sections? Does anyone have an alternative system? I could sure use some input or ideas anyone welcome
> and most appreciated.
> 
> Maria Mejia
> Lead Histologist
> UCSF
> Mission Bay
> San Francisco, CA
> 
> 
> 
> 
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