FW: Antigen Retreival Solution

From:Phyllis Davie <pdavie@phenopath.com>


From: Phyllis Davie <pdavie@phenopath.com>
Date: Fri, 02 Mar 2001 11:54:54 -0800
To: Gerard Spoelstra <spoelstr@numbat.murdoch.edu.au>,
<histonet@pathology.swmed.edu>
Subject: Re: Antigen Retreival Solution

Enclosed please find our methods for antigen retrieval.   We use several
different methods.  Please feel free to contact me if you have any
questions.
Best of luck,

Phyllis Davie
PhenoPath Laboratories--Seattle, WA
pdavie@phenopath.com

PRETREATMENT, ANTIGEN RETRIEVAL


HEAT INDUCED EPITOPE RETRIEVAL (HIER)

Purpose:  The Heat Induced Epitope Retrieval (HIER) procedure is used to
unmask or retrieve epitopes of poorly fixed or over-fixed tissues, as well
as unmask epitopes in previously non-reactive tissue.  It is especially
useful in formalin fixed tissues.  Formalin fixes tissues by blocking amido
groups and by forming methylene bridges between several amino acids in
polypeptides.  By this "cross-linking" mechanism, formalin effects a
variable number of chemical links not only within a protein molecule but
also with adjacent proteins.  These cross-linked proteins are believed to
block access of antibodies to their target epitopes, a process often called
"masking" of antigens.  Although the exact mechanism by which HIER unmasks
epitopes is unknown, many studies have demonstrated improved
immunoreactivity of tissue sections following HIER.

A variety of heating techniques are acceptable for the performance of HIER,
including microwave, pressure cooker, hot plates, dry ovens, autoclaves, and
steamers.  In this procedure, we will describe the three heating techniques
used in this laboratory.

In addition, a large variety of buffer types and conditions (concentration,
pHŠ) have been successfully employed in HIER.  Again, we will describe those
buffers actually in use in this laboratory.  Determination of which antigen
retrieval technique is used for a particular antibody is arrived at
empirically, and recorded in the Antibody List (FileMaker Pro database).

Buffers:

I.    Citrate Buffer pH 6
Reagents
a.    Citric Acid, monohydrate, granular
    Baker Analyzed Reagent
    J.T. Baker Inc., Phillipsburg, NJ 08865
    Catalog #0110-01
    Storage: Room Temperature
b.    Filtered distilled water
c.    10M Sodium Hydroxide
    Anderson Laboratories, Inc., Fort Worth, TX 76112
    Catalog # 65550
    Storage: Room Temperature in the base cupboard
Preparation:    10 mM Citrate Buffer
    a.     Dissolve 8.41g citric acid monohydrate in 4L of dH20
    b.    pH to 6.0 +/- 0.3 with 10M NaOH
Storage:  Room Temperature
Shelf life:  3 months


 II.    EDTA, 0.01M (= 10mM), pH 8.0
Reagents:
a) EDTA, disodium salt
    Storage:  room temperature
    Sigma Chem. Co., St. Louis, MO 63178
    Cat.#  ED2SS
b)    filtered deionized water
c)    10M Sodium Hydroxide (NaOH)
    Anderson Laboratories, Inc., Fort Worth, TX 76112
    Catalog # 65550
    Storage: Room Temperature in the base cupboard
Preparation:
1.    Dissolve:
     0.37g    EDTA
    1.0 L    diH2O
2.    Adjust pH to 8.0 with NaOH.  Adjust pH very carefully.  It can zoom
off into the distance as you approach 8.
Storage:  Room Temperature
Shelf life:  2 weeks


III.    Tris Buffer, 0.5M, pH 10 (10x more concentrated than standard)
Reagents:  
a.    Trizma base (Tris[hydroxymethyl]aminomethane) (C4H11NO3)
    Reagent Grade
    Storage:  Room temperature
    Sigma Chemical Company  St. Louis, MO  63178
    Catalog # T-1503
b.    Filtered distilled water
c.    Hydrochloric acid, concentrated (HCl approximately 37%)
    AR Select
    Storage:  Room temperature
    Mallinckrodt Inc.  Paris, KY  40361
    Catalog # 5587-03
Preparation 0.5M pH10 Tris:
a.    Dissolve 242.20 g Trizma Base in about 3800 ml distilled water.
b.    Adjust pH to 10.0 +/- 0.1 with concentrated HCl.  QS to 4L.
Storage:  Refrigerated (4-8o C)
Shelf Life:  2 months


IV. UREA, 3M
Purpose:  Used in conjunction with the microwave to reveal/retrieve
antigens.  It may also enhance reactivity of poorly fixed tissues, as well
as unmask antigens in previously non-reactive tissue.
Reagents:
a.    Urea, ACS reagent, CH4N2O
Sigma Chemical Co., St. Louis, MO 63178
Catalog # U-5128
Storage:  room temperature
b.    Filtered deionized water
Preparation, 3M Urea;
1.    Dissolve 540g urea in 4L of dH2O.  (weigh and mix in a hood, wearing
appropriate protective clothing)
    Storage:  room temperature
    Shelf Life:  3 months
Other Supplies:  Microwave equipped with an exhaust system.
CAUTION:      Urea is a health hazard.  Harmful by inhalation, in contact
with skin, and if swallowed.  Mechanical exhaust required.  Irritating to
eyes, respiratory system and skin.  Possible risk of irreversible effects.
Possible mutagen, may cause abortion.  In case of contact with eyes, rinse
immediately with plenty of water and seek medical advice.  Wear suitable
protective clothing.  Do not breathe dust.  Refer to MSDS for further
information.


Procedures  (any buffer):

Variation 1:  Microwave (Open Boat) Heat Induced Epitope Retrieval
a.    Place slides in a plastic rack (no metal).  If there are not enough
slides to fill the rack, then fill the rack with blank slides.
b.    After deparaffinization and rehydration, wash the slides in dH20.
c.    Place the slides in 200 ml of desired buffer, cover  with a lid
loosely and place in microwave for pre-determined length of time.    Do not
let fluid level evaporate below level of tissue -- stop microwave every 4-5
minutes and add diH2O.
Before starting microwave, fill glass tray with distilled water to prevent
burning of spilled buffers
d.    Remove carefully from microwave.  Let slides sit in the buffer at room
temperature for 20 minutes, wash in PBS and proceed with immunoperoxidase
procedure. NOTE: This "cool down" period has been determined to be a crucial
step in this procedure.  Do not skip this step.

Variation 2:  Microwave Pressure Cooker Heat Induced Epitope Retrieval
a. A NordicWare 2.5-quart microwave pressure cooker is loaded with 1200 ml
of 6 mM Citrate pH 6.0 at room temperature.  To ensure standardization, 4
full racks of slides are loaded into the pressure cooker with each run; if
insufficient test slides are available, the remainder of slides in the racks
will be dummy slides (blank glass).
b. Set the microwave power level to full, and the timer to 30 minutes.
Place the loaded and sealed pressure cooker in the microwave, making sure
the red weight is in place, and start the microwave.
c. Listen for the sound of steam escaping the microwave pressure cooker.
When steam is heard, stop the microwave, and reset the timer for the
indicated amount of time (e.g. 10 minutes, 15 minutesŠ a separate pressure
cooker run is needed for each pretreatment time, since slides cannot be
removed from the pressure cooker partway through.)
d. At the end of the set time, open the microwave, and remove the pressure
cooker; being careful of the heat, and making sure that the red weight is
NOT knocked off.  (This would allow all the steam to rapidly escape,
possibly burning the operator, and almost certainly drying the slides, which
is usually detrimental{i.e. ruinous}.)
e. Set a timer for 10 minutes to allow the pressure cooker to slowly cool
down and depressurize.  The yellow tab indicating pressure should drop down.
At this time, remove the red weight carefully, and allow the remaining steam
to vent, then remove the lid, and check the temperature.  The expected
temperature is around 90 degrees C.  Next, cool the slides completely by
adding water to the pressure cooker.

Variation 3: Steamer Heat Induced Epitope Retrieval
a. Preheat the steamer by filling the steamer base with distilled water to
the highest fill line, and turning the unit on.  Set the timer to maximum.
Cover the steaming bowl with the lid, and place on the base.  Do not use the
drip tray. 
b. Preheat approximately 200 ml of desired buffer to at least 90šC by
placing it in the steamer for approximately 20 minutes prior to use.  It is
fine if hotter than 90šC, but it must not be any cooler.  (Alternately,
preheat buffer(s) in the microwave to over 90šC, and transfer to the
steamer.)
c. Place slides in a rack.
d. After deparaffinization and rehydration, wash the slides in dH2O.
e. Place the slide rack in the preheated container of buffer, cover with a
lid loosely (optional), and place in the heated steamer for the
pre-determined amount of time.  (usually 20 minutes). Set a separate timer
for each duration of treatment desired.  When the timer alarms, remove the
slides that are ready from the steamer and quickly replace the lid so that a
minimum of heat is lost. Continue timing any slides which require longer
treatment and set a timer to measure the 20 minute cool down period for the
slides which have already been removed.
f. When all slides have been treated, unplug the steamer and decant the
remaining water into the sink.
g. Pick up all the slides and wash in one change of PBS and one change of
PBS with BSA/Triton, then a fresh change of PBS for a total of 5 minutes.
Proceed with antibody application.
Procedure Notes:  
1. Be careful to lift the lid by tilting it away from your face to avoid a
rush of steam in your face.
2. Do not rely on the steamer dial as it is not a reliable timer for this
purpose.
3. .If using Urea, the steamer should be in a hood to avoid exposure to urea
fumes.





    References:  
1.    Battifora H, Alsabeh R, Jenkins KA, Gown AM:  "Epitope Retrieval
(Unmasking) in Immunohistochemsitry".  Advances in Pathology and Laboratory
Medicine, vol. 8, 101-118, 1995
2.    Gown AM, De Wever N, Battifora H: "Microwave-Based Antigenic
Unmasking: A Revolutionary New Technique for Routine Immunohistochemsitry"
Appl Immunohistochem 1(4): 256-266, 1993.
3.    Taylor CR, Shi SR, Chen C, Young L, Yang C, Cote RJ:  "Comparative
Study of Antigen Retrieval Heating Medhods:  Microwave, Microwave and
Pressure Cooker, Autoclave, and Steamer"  Biotechnic & Histochemistry 71(5):
263-270, 1996.
4.    Shi SR, Cote RF, Young L, Imam SA, Taylor CR:  "Use of pH 9.5 Tris-HCl
Buffer Containing 5% Urea for Antigen Retrieval Immunohistochemistry"
Biotechnic & Histochemistry 71(4): 190-196, 1996.

---------------------


> From: Gerard Spoelstra <spoelstr@numbat.murdoch.edu.au>
> Date: Fri, 02 Mar 2001 09:59:32 +0800
> To: histonet@pathology.swmed.edu
> Subject: Antigen Retreival Solution
> 
> Dear Histoneters, For those of you who make up your own, "Antigen Retreival
> Solution" could I please have the method for making it up. Thanks in advance.
> Gerard Spoelstra
> Medical Scientist
> School of Biomedical Science
> Murdoch University
> Western Australia
> 
> 




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