RE: [Histonet] Immunoflouresence Questions

From:"Houston, Ronnie" (by way of histonet)


For those unfamiliar with Mowiol, it is a Polyvinyl Alcohol and can be
obtained from Calbiochem.


As Tim said, DABCO can be added as an antifading agent (25mg/ml), as can
para-phenylene diamine (10mg/ml) and n-propyl gallate (2% w/v); in fact all
three agents can be combined in one solution. All these substances are
harmful by ingestion, inhalation and skin-absorption.

In my opinion, the Alexa dyes from Molecular Probes are much more resistant
to photo bleaching than any others on the market, and if memory serves me
correctly Molecular Probes do have a commercial mounting medium that
contains a reagent (proprietary) to prevent fading.

Ronnie

Ronnie Houston
Director of Anatomic Pathology
Bon Secours HealthPartners Laboratories
5801 Bremo Road
Richmond, VA 23226
(804) 2877972
(804) 2877906 - fax
ronnie_houston@bshsi.com





-----Original Message-----
From: ana.merino-trigo [mailto:ana.merino-trigo@wanadoo.fr]
Sent: Thursday, October 13, 2005 3:49 PM
To: Rene J Buesa; Nita Searcy; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Immunoflouresence Questions


Hi,


I'm not really an expertise, but in my first postdoc in Australia, I was
working in a Cell biology department and we were using Mowiol as mounting
medium. We kept the slides in dark at 4C, and the fluorescence was the same
the day after, than the week after, even I was reviewing slides after one
month on the fridge, with no a great difference. However, I was working with
cell lines, using as FITC, TxRed, Alexa ... antibodies and I don't really
now if tissues fades quicker. Attached the protocol used to prepare Mowiol.


I do want to take the opportunity to say everyone thank you, I'm learning a
lot by reading Histonet disccusions. Great work everyone.


Mowiol Mounting Medium pH 8.5



Materials




Hopval 5-88                             4.8g

glycerol                                   12.0g

MilliQ H2O                              12.0ml

0.2M Tris-HCl pH 8.5              24.0ml




Tris                                         2.42g Tris base

milliQ H2O                               100ml

pH to 8.5 with 6N HCl




Method




.     Place glycerol in 50ml disposable blue cap tube

.     Add Hopval and stir thoroughly

.     Add H2O and leave for 2h at R.T.

.     Add tris and incubate at ~53C until the Hopval has dissolved,
stirring occassionally (takes ages)

.     clarify by centrifugation at 3500rpm for 30min and aliquot the
supernatant into 1ml in 1.5 ml tubes

.     store at -20C, stable at this temp for 12 months

.     once defrosted, stable at R.T. for one month




Thanks

Ana



  ----- Original Message -----

  From: Rene J Buesa

  To: Nita Searcy ; histonet@lists.utsouthwestern.edu

  Sent: Thursday, October 13, 2005 9:28 PM
  Subject: Re: [Histonet] Immunoflouresence Questions


  Nita:
  You are right, immunofluorescence procedures will fade, and fast; that is
the reason behind taking photographs of the results.
  You may keep the slides in a refrigerator and will be able to register
immunofluorescence again, but WEAKER, the next day.
  How long will this fluorescence will remain visible in refrigerated slides
depends on the case, but cannot be predicted in general. When the
pathologist that is scheduled to read our procedures is not available, we
keep the slides for the next day, but that is all; no permanent solution.
  There is no media that will retard fading (is something inherent to the
FITC conjugated antibody and has nothing to do with the medium.
  Probably your clinician "heard" something but evidently does not know the
procedural details, or he may know something that neither you or I know.
  Rene J.

  Nita Searcy  wrote:
  I have a tech that is asking these questions regarding possible "new
  procedures". We have a new clinician that has "new" ideas.

  1. Does storing slides in refrigerator after staining aid in anyway? (I
  had never seen this before ; always stored in dark @ room temperature).

  2. IS there a mounting media that aids in permanence?


  3. To my knowledge, these slides are not permanent (no matter what you
  do) but will inspectors really expect to see them- even in a faded
  condition? (we take pictures of the kidneys)

  Thanks

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