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

[Maria Mejia via Histonet]

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 <rjbu...@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@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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> 
> 

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

[Maria Mejia via Histonet]

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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[Histonet] need automation system for fluorescent Multiplex IHC

[Maria Mejia via Histonet]

Dear All,

I work in a research lab where the focus is on early stages of Alzheimer’s 
Disease.
We are seriously considering using an automated staining system for fluorescent 
Multiplex
IHC, for the simultaneous detection of multiple (2-6 or more) proteins of 
interest in 
FFPE brain sections.

However, our lab has never used an automated staining system before, so we lack
the abilities and experience to make the correct choice for our lab’s needs.

Cost; the price of a USED system, since we are a research lab since a new 
system is more
expensive, user friendly & size of system are of course major considerations as 
well 
as the consumables that go along with the system e.g. reagents, containers, & 
anything else
to consider as well.

Please, I’m reaching out to anyone (system users & vendors) who can help our 
lab make a smart choice.
I would be most grateful for any recommendations or suggestions. I very much 
look forward to hearing
from you.

Best
Maria Mejia
Lead Histologist
UCSF 
Memory & Aging Center


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Re: [Histonet] Unprocessing tissue from paraffin

[Maria Mejia via Histonet]

Hi Millis,

Lets see if I’m understanding you correctly. 
1) You have unprocessed tissue. What kind of unprocessed tissue & how thick are 
the free-floating sections?
2) Are the tissue fixed? If so, with what? & For how long?
3) You want to stain the unknown whole mount using various histochemical stains 
or IHC stains?
4) Can you please explain why after staining you need to reprocess for 
paraffin? 
Cause once these mystery sections are processed you'll have to cut them using a 
rotary microtome.

I can maybe help you, but your going to have to provide more detail 
information. Also provide your
processing protocol.

Regards
Maria Mejia
Lead Histologist
Memory & Aging Center
UCSF
> On Sep 21, 2016, at 12:38 PM, Caroline Miller via Histonet 
>  wrote:
> 
> Hi there Histonetters!
> 
> I am currently working through some issues of unprocessing tissue to then
> restain 'whole mount' with various stains, and then reprocess back to
> paraffin.
> 
> I am running into issues of tissue being brittle and scratchy tissue after
> reprocessing and sectioning.
> 
> Can anyone offer any advice on kinder dewaxing procedures and / or
> additives to the unprocessing / processing fluids that might help in the
> resulting piece of tissue?
> 
> I have currently been putting it through the cleaning cycle on the
> processor, so standard xylene then alcohol treatment.
> 
> thanks in advance for your advice!
> mills
> 
> 
> 
> 
> -- 
> Caroline Miller (mills)
> Director of Histology
> 3Scan.com
> 415 2187297
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[Histonet] slide labeler advice for research lab

[Maria Mejia via Histonet]

Our research lab is in the market for a slide labeler. We are an IHC research 
lab (NOT clinical) working on both
paraffin & thick free-floating human whole brain & brainstem sections. Since 
the lab has never used a 
slide labeler before (we’ve been manually making our labels), the labeler 
should be easy to use, so 
no one has to spend a lot of time figuring & programming the unit. Since we use 
paraffin standard glass
slides as well as 2 inche x 3 inch glass slides, we need a labeler that we 
could create formats for different sample
sizes and types e.g. ID #, Casp 6 + TH IHC + (name of counterstain used) & 
date..etc.
The unit should also have barcoding capabilities.

Any advice or suggestions are appreciated. Vendors welcome also.

Maria Mejia
Lead Histologist
Memory & Aging Center
UCSF
SF CA
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[Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC!

[Maria Mejia via Histonet]

Happy New Year Everyone,

I'm the lead histologist working in an IHC based research lab focused on early 
stages
of Alzheimer's disease.  We work on paraffin sections processed & cut from 
600um celloidin 
sections.  Including a lot of 60um cellodin sections from whole human 
brainstem.  

For years, everything has going good regarding counterstaining after single & 
double IHC staining
on 60um free-floating sections.  However for the past two months we've 
struggled to achieve
good visible counterstaining on IHC sections to count the stained neurons - to 
see clearly
the nucleus & nucleolus!  

For a number of years, Gallocyanine was our choice of counterstain after IHC.  
Now, it's
NOT working (neurons not stained visible enough to count).  We've also tried 
cresyl violet
counterstain -  staining too weak!   In both counterstains, we modified
the staining protocols quite a number of times to get good visible staining - 
nothing!!!

Strangle because we get lovely counterstained neurons with NO IHC staining on 
our 60um
sections, but as soon as we take the sections through the IHC protocol e.g. 
antigen retrieval, 
antibodies & chromogens - counterstain is too weak!  Our paraffin IHC sections 
work & look 
wonderful!

Now, my PI wants to try methyl green counterstain, however I think we'll have 
the same problem.
Here's what I need help with:

1) Can someone please explain the reason or theory  behind the failure of 
counterstain uptake
by cells such as human neurons on 60um celloidin sections?

2) Can anyone please offer staining protocols that use alternative dehydration 
& clearing reagents.
I've been using alcohols dehydration (96% & 100%) without success as well as 
clearing with xylene - 
which hardens the tissue if left too long in this reagent. 

 I was thinking of perhaps using acetone instead of alcohols & maybe using a 
methyl salicylate or
chloroform.  Thoughts anyone?

I wish Dr Chris van der Loos was still with us.  I'd dearly like to hear from 
anyone who can help with
this issue - Gayle Callis, Terry Johnson, Dr Hohn Kiernan et al.

Any assistance anyone can provide will be greatly appreciated!

Best
Maria Mejia
UCSF
Memory & Aging Department
San Francisco, CA
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