Re: [Histonet] X-gal staining

2020-01-12 Thread H. Etchevers via Histonet 
I agree with Carl about the X-gal activity being destroyed by processing 
for paraffin sections (I'm a researcher, not a histologist).


However, in our hands the anti-b-galactosidase antibody, reference 
A-11132, from ThermoFisher DOES work on paraffin sections (many others 
don't). It has been published with subsequent incubation with a 
biotinylated secondary (Vector BA-1000) and standard procedures 
thereafter to show good signal with DAB. We do immunofluorescence.


MPBio used to sell a good polyclonal for this purpose, but it has since 
been discontinued.


Best wishes,

Heather

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Re: [Histonet] X-gal staining

2020-01-11 Thread Paula Keene Pierce via Histonet 
I have done paraffin processing and sectioning of X Gal stained eyes and embryo 
heads.
The staining is done for the blue color first, post fixed, and then 
processed.Xylene should be avoided, as it is what removes the X Gal target, and 
a xylene substitute used such as StatLab's XS3.
Deparaffinization and dehydration for coverslipping should also be done with 
the xylene substitute.
I counterstain with Nuclear Fast Red.
Paula Keene Pierce, BS, HTL(ASCP)HTPresidentExcalibur Pathology, Inc.5830 N 
Blue Lake DriveNorman, OK 73069PH 405-759-3953http://www.excaliburpathology.com

A sharp knife is nothing without a sharp eye. - Klingon Proverb 

On Saturday, January 11, 2020, 01:22:43 PM CST, Hobbs, Carl via Histonet 
 wrote:  
 
 Hi
No replies so far so.my pennyworth.
Imho ...no
beta Gal enzyme is inactivated by std Pwax processing.
So, when you apply the X-gal substrate and chromogen solution to Pwax sections, 
 it will not give you a positive blue final reaction product
If you want to detect beta Gal in FFPWS and visualise with DAB,  you will have 
to use an anti beta Gal antibody.
Finding an anti beta Gal enzyme antibody, that works in FFPWS +/- AR is not so 
easy.

Good luck

Carl

Carl Hobbs FIBMS
Histology and Imaging Manager
Wolfson CARD
Guys Campus, London Bridge 
Kings College London
London
SE1 1UL
 

020 7848 6813






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Today's Topics:


  1. X-gal staining (Margaryan, Naira)

  2. (no subject) (Waitts, Celeste)



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Message: 1

Date: Tue, 7 Jan 2020 18:21:31 +

From: "Margaryan, Naira" 

To: "'histonet-request (histonet@lists.utsouthwestern.edu)'"

        

Subject: [Histonet] X-gal staining

Message-ID:

        


        

Content-Type: text/plain; charset="us-ascii"


Hello histonetters,


I have request from a scientist to perform X-gal staining on the formalin fixed 
paraffin-embedded kidney.

I was wondering if there is any possibility to do this staining on FFPE tissue 
as well as if there any possibility to perform a peroxidase DAB  staining for 
the X-gal? If so, may I ask you for a detailed protocol to perform this 
staining.

Any input and explanation why it is impossible is appreciated.



Thanks in advance,

Naira




--


Message: 2

Date: Wed, 8 Jan 2020 16:46:18 +

From: "Waitts, Celeste" 

To: "'histonet@lists.utsouthwestern.edu'"

        

Subject: [Histonet] (no subject)

Message-ID:

        <131bf77daa5d4ba08f9076d9afbf1...@exchnode1.local.centrastate.com>

Content-Type: text/plain; charset="us-ascii"


Question      VIP6    SHORT BIOPSY RUN    USING CLEARITE

ANYTHING


Celeste Waitts

Histology Supervisor

Centrastate Medical Center

Freehold, NJ  07728

(732-303-5071)




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Re: [Histonet] X-gal staining

2020-01-11 Thread Hobbs, Carl via Histonet 
Hi
No replies so far so.my pennyworth.
Imho ...no
beta Gal enzyme is inactivated by std Pwax processing.
So, when you apply the X-gal substrate and chromogen solution to Pwax sections, 
 it will not give you a positive blue final reaction product
If you want to detect beta Gal in FFPWS and visualise with DAB,  you will have 
to use an anti beta Gal antibody.
Finding an anti beta Gal enzyme antibody, that works in FFPWS +/- AR is not so 
easy.

Good luck

Carl

Carl Hobbs FIBMS
Histology and Imaging Manager
Wolfson CARD
Guys Campus, London Bridge 
Kings College London
London
SE1 1UL
 

020 7848 6813






From: histonet-requ...@lists.utsouthwestern.edu 


Sent: 08 January 2020 18:00

To: histonet@lists.utsouthwestern.edu 

Subject: Histonet Digest, Vol 194, Issue 4
 

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Today's Topics:


   1. X-gal staining (Margaryan, Naira)

   2. (no subject) (Waitts, Celeste)



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Message: 1

Date: Tue, 7 Jan 2020 18:21:31 +

From: "Margaryan, Naira" 

To: "'histonet-request (histonet@lists.utsouthwestern.edu)'"



Subject: [Histonet] X-gal staining

Message-ID:






Content-Type: text/plain; charset="us-ascii"


Hello histonetters,


I have request from a scientist to perform X-gal staining on the formalin fixed 
paraffin-embedded kidney.

I was wondering if there is any possibility to do this staining on FFPE tissue 
as well as if there any possibility to perform a peroxidase DAB  staining for 
the X-gal? If so, may I ask you for a detailed protocol to perform this 
staining.

Any input and explanation why it is impossible is appreciated.



Thanks in advance,

Naira




--


Message: 2

Date: Wed, 8 Jan 2020 16:46:18 +

From: "Waitts, Celeste" 

To: "'histonet@lists.utsouthwestern.edu'"



Subject: [Histonet] (no subject)

Message-ID:

<131bf77daa5d4ba08f9076d9afbf1...@exchnode1.local.centrastate.com>

Content-Type: text/plain; charset="us-ascii"


Question   VIP6SHORT BIOPSY RUNUSING CLEARITE

ANYTHING


Celeste Waitts

Histology Supervisor

Centrastate Medical Center

Freehold, NJ  07728

(732-303-5071)




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Re: [Histonet] X-gal staining

2015-01-08 Thread koellingr 
Kristopher, 
beta-gal is an enzyme and as far as I know is rendered inert after FFPE.  I did 
do fresh skin in X-gal to target, as you would with any whole mount specimen, 
THEN FFPE and cut sections to it to see the signal.  If that is not an option, 
go after it with an anti-beta galactosidase antibody (polyclonal-like Pierce I 
used-have zero relationship to them) from several places.  Make sure the 
immunogen is a full, length protein that produced the antibody, and it will go 
much easier. 
  
Ray 
  
Raymond Koelling 
Seattle, WA area 

- Original Message -

From: Kristopher Kalleberg kristopher.kalleb...@unilever.com 
To: histonet-requ...@lists.utsouthwestern.edu, 
histonet@lists.utsouthwestern.edu 
Sent: Thursday, January 8, 2015 9:53:38 AM 
Subject: [Histonet] X-gal staining 

Hello All, 

Does anyone know of a commercially available X-gal stain that would work on 
formalin fixed paraffin embedded skin samples in order to detect beta 
galactosidase.  Thank you in advance. 

Kris 
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Re: [Histonet] X-gal staining

2009-08-16 Thread John Kiernan 
Dear ndev...@stanford.edu
 
You may be detecting endogenous animal in addition to bacterial (lac-z) 
beta-galactosidase activity. Indigogenic methods were recently reviewed in 
Biotechnic  Histochemistry 82: 72-103 (2007). The pH of the indigogenic 
substrate solution is important. Many other factors can cause false positive 
and false negative X-gal staining.  Do your homework!
 
John Kiernan
Anatomy, UWO
London, Canada
= = =
- Original Message -
From: Nicholas David Evans ndev...@stanford.edu
Date: Thursday, August 13, 2009 15:51
Subject: [Histonet] X-gal staining
To: histonet@lists.utsouthwestern.edu

 Hello,
 
 I was wondering whether anyone might advise me on X-gal 
 staining. 
 
 I am looking at the expression of LacZ knocked into the locus of 
 our gene
 of interest. We cryoembed, take sections and stain for beta-
 galactosidaseactivity using X-gal. The blue stain is often 
 punctuate and noticeably
 limited to the nucleus, whereas in other areas the staining is 
 diffuse and
 appears throughout the cytoplasm of the cells. I have guessed 
 that this
 may be related to the level of expression; i.e. that in cells 
 with a low
 reporter activity beta-gal is limited to the nucleus, but that 
 at higher
 expression the whole cell becomes riddled with it. If anyone knows
 anything about the subcellular distribution/localisation of beta-
 gal in
 mammalian cells, or can direct me to some good literature I 
 would be
 grateful.
 
 Best wishes
 Nick
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