[Histonet] Use of sodium borohydride in immunofluorescence

[Lucie Guernsey via Histonet]

Hi,

I'm currently going over IHC SOPs that were written by lab members that
have long ago moved on to other jobs. One inconsistency that I've come
across is the use of sodium borohydride in immunofluorescence.

Our protocols state that a '0.5% sodium borohydride solution should be used
to block aldehyde fluorescence if staining CNS tissue in paraffin sections'
(this step is not included in our cryosection protocol).

Is there a particular reason that it should only be done in CNS tissue?
Seems to me that all tissues, regardless of type, should benefit from
blocking aldehyde fluorescence as the problem is caused by the fixative,
not the tissue type. Which leads me to the follow up question of: Our
cryosection protocol should also include sodium borohydride blocking if the
tissues were at any point fixed using an aldehyde fixative, correct?

There's a chance I have pregnancy brain and an obvious answer is slipping
past me, but my current opinion is that any and all immunofluorescence done
on paraformaldehyde (our usual fixative) fixed tissue should undergo sodium
borohydride blocking. Am I missing something?

Thanks for any help in clarifying this!

Lucie

Lucie Guernsey
UC San Diego
lguern...@ucsd.edu
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[Histonet] FT EVENING HT POSTITION Delray Beach, FL

[Kari Simeone via Histonet]

Hi Histonetters! We are looking for a full time licensed HISTOTECHNOLOGIST here 
in our very busy Delray Beach Florida Dermatology Lab. This is a permanent full 
time EVENING SHIFT (40 hours) position with benefits (medical/401k/vacation). 
THIS IS A DRUG FREE WORKPLACE. Background check, personality test and drug test 
will be necessary. Sorry, no relocation assistance provided.


***PLEASE NO HEAD HUNTERS/PLACEMENT SERVICES***!!!

Please fill out employment application HERE
https://www.indeed.com/viewjob?jk=ec6f15bf3fbbbdf7=histotechnologist=florida=1bbmf648ua3cqdc2=web
^^you MUST follow this application link to apply! No exceptions.

*FULL TIME position Mon-Fri OR Sun-Thurs 5P-1:30AM (START TIME CAN VARY FROM 
5-10P)
*MUST be licensed as a FL HISTOTECHNOLOGIST ONLY (will be working solo some of 
your shift)
*MUST have at LEAST FIVE (5) years experience (dermatology preferred)
 Please DO NOT respond if no EXPERIENCE!
*VERY proficient in embedding and microtomy
*WILL MOSTLY BE EMBEDDING EXCISION BLOCKS, please know DERMS
*must be self motivated, reliable and a team player
*knowledge in operating Ventana and Leica equipment desired (not necessary)
*some IHC experience preferred but not necessary



Kari M Simeone




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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  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 
>  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] Toxicology Histology Supervisor Position

[Oconnor, Jackie M via Histonet]

Hello Histonetters - we are looking for a Histology Supervisor.  If you are 
interested, please email me.
Jackie
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Re: [Histonet] need help staining 120um human whole brain sections!

[Rene J Buesa via Histonet]

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 
 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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