Aptum 2100 Retriever User manual

Retriever 2100
OPERATION MANUAL
Since 2007
Unique technology to retrieve the antigens on fixed,
paraffin-embedded tissue sections
US Patent Pending 2007 0227298A1

2
INTRODUCTION
INTRODUCTIONINTRODUCTION
INTRODUCTION
2100
21002100
2100-
--
-Retriever
RetrieverRetriever
Retriever, or, from now on simply Retriever, is a benc -top model for
t ermally processing slides of t e formalin-fixed, paraffin embedded tis-
sues prior to immunostaining. T e model is designed to ensure identical
processing of all t e samples during a processing cycle, as well as t e
identical processing of t e samples between individual sessions.
T e Retriever is developed on t e basis of an autoclave. T ere ave been
earlier reports t at autoclaving formalin-fixed sections may be in no way
less effective t an processing t em in a microwave. After a year of testing
we found t e optimal configuration of suc an autoclave. T e core princi-
ple is t e eating of t e c ambers wit t e slides at ig temperature
(>120
0
C) and ig pressure. T e c ambers reac t e required tempera-
ture and remain t ere for a fixed time. T e specially designed t ermal
walls of t e unit control t e cooling rate of t e inner c amber and slides.
T e unit is fully automatic.
Reproducibility
ReproducibilityReproducibility
Reproducibility. T ere is no boiling of t e buffer, and no cold zone effect,
usual to a microwave, w en some of t e sections are eated less t an
ot ers. All sections are processed uniformly and always for t e same
lengt of time. T ere are no variations from cycle to cycle so you can
compare t e sections processed wit a gap of a couple of mont s (or
years). Since t e cycle is fully automatic, t e eating of t e sections, ex-
posure to ig pressure and temperature, and even cooling (also quite
important) are standardized. So are your results.
Quality of t e results
Quality of t e resultsQuality of t e results
Quality of t e results.
..
. T e Retriever preserves t e tissue morp ology,
t us your images will be of muc ig er quality. For some antigens we
found t at t e intensity of t e staining is muc ig er t an wit standard
met ods (especially for nuclear antigens, suc as estrogen receptor).
Your time
Your timeYour time
Your time. All you ave to do is to put slides in a c amber and pus t e
button. W ile t e unit is working, you do not ave to be nearby, you can
carry on wit ot er matters.
All antibodies t at can be used on microwave treated tissues can be also
used in Retriever.
WHAT IS THE RETRIEVER
HOW DOES IT WORK
ADVATAGES OVER MICROVAWE

3
CONTENT
CONTENTCONTENT
CONTENT
Retriever and Acessories (Sc eme)
4
Getting Started
5
Fixing Tissue Samples
6
Cuttung Tissue Sections
7
Dewaxing Tissue Sections
8
C oosing t e Buffer
9
Processing Sections in Retriever
10
Immunostaining
12
Examples of Staining
13
Do’s and Don’t’s
14
Care and Maintanance
16
Troubles ooting
17
Safety Features of Retriever
18
Certificates of Retriever
19
Tec nical Specifications
20

4
A
B
D
E
F
C
G
L1
L2 L3 L4 L5
A
B
C
D
E
F
G
Depressurization Valve
Display Panel
Pressure Rize Indicator
Body of Retriever
Slide Cha ber
Cha ber Rack
Display Lights
L1- Power on -
L2- Heating on -
L3 - The cycle conditions are reached
-
L4- Cycle co plete -
L5 - Fault Light -
YOUR RETRIEVER
YOUR RETRIEVERYOUR RETRIEVER
YOUR RETRIEVER
T e supplier Retriever set contains t e processing unit, slide c amber
(allows to fit 18 Slides) and C amber Rack for 6 Slide C ambers (108
slides in total). On t e control panel t e Start Button and indicator lig ts
are located t at s ow different stages of t e processing program.
Please get acquainted wit t e location of various parts of Retriever and
t e signals displayed by indicator lig ts,

5
Retriever is an autoclave
Retriever is an autoclave Retriever is an autoclave
Retriever is an autoclave and t us as to be andled appropriately and
wit care.
Place t e Retriever on t e benc -top at t e open space since some vapor
will be coming out of t e pressure indicator. Please use t e appropriate
voltage outlet if your laboratory as bot 110-115V and 220V. All models
for US and Japan are standardly s ipped as 110V models, unless specifi-
cally requested ot erwise.
WARNING! Mains outlet MUST BE EARTHED (GROUNDED)
WARNING! Mains outlet MUST BE EARTHED (GROUNDED) WARNING! Mains outlet MUST BE EARTHED (GROUNDED)
WARNING! Mains outlet MUST BE EARTHED (GROUNDED)
T e mains plug s ould always be easily accessible as it is to be relied
upon as "t e means of disconnection".
GETTING STARTED
GETTING STARTEDGETTING STARTED
GETTING STARTED
Before using t e Retriever for t e first time please take time to read
Before using t e Retriever for t e first time please take time to read Before using t e Retriever for t e first time please take time to read
Before using t e Retriever for t e first time please take time to read
t e following pages to familiarize yourself wit t e operation of t e unit.
t e following pages to familiarize yourself wit t e operation of t e unit. t e following pages to familiarize yourself wit t e operation of t e unit.
t e following pages to familiarize yourself wit t e operation of t e unit.
We strongly recommend t at all users of t e Retriever are trained in its
We strongly recommend t at all users of t e Retriever are trained in its We strongly recommend t at all users of t e Retriever are trained in its
We strongly recommend t at all users of t e Retriever are trained in its
operation.
operation. operation.
operation.
Please notice t e zones (as indicated above) of t e eat coming out of
t e unit. W en cycle is run, t e Body may be ot. Some ot steam may be
released from t e site of Pressure indicator and Depressurization Valve.
WARNING! T e Depressurization Valve must be closed before t e unit is
started for t e cycle running!
Open
Closed
C
B
D
!
!
!
Warning:
Hot Stea
Warning:
May be Hot
Caution:
Electric Shock Hazard Body
Control Panel
A
Pressure
Indicator
Depressurization
Valve

6
We ave developed t e Retriever for formalin fixed, paraffin embedded
tissues, t e most routine material available from tissue collections and
easy to prepare from fres tissue samples. A routine protocol used in t e
majority of pat ology laboratories is sufficient. Your approximate protocol
s ould look like t is:
NOTES
NOTESNOTES
NOTES
 W en you are preparing t e specimen yourself, please was all resid-
ual fixative properly by extended incubation in 50% and 70% et anol
prior to submitting it to furt er de ydration and embedding to paraffin
 Isolating tissue specimen please avoid tissue autolysis. To prevent t is
t e best solution is to place t e tissues straig t to t e cold fixative.
Alternatively use specialized solutions for transporting tissues. Use a
cold plate to andle t e tissues w enever possible.
 Consider t at very ard tissues may require an extended (up to 48
ours) fixation time
 To preserve good infrastructure of certain tissues suc as lung, pan-
creas, liver, a fixative perfusion is required. Alt oug t is may not be
necessary for most immuno istology stainings, it may be desirable in
cases w en ultrastructure of a particular organ area is of importance.
If t e tissues ave to be compared wit respect to expression of a cer-
tain antigen, please control t at t ey all undergo identical fixation
(w ic includes size of samples, time of incubation).
FIXING TISSUE SAMPLES
FIXING TISSUE SAMPLESFIXING TISSUE SAMPLES
FIXING TISSUE SAMPLES
PROTOCOL R1
 Cut tissue into 4 to 7 mm blocks
Cut tissue into 4 to 7 mm blocksCut tissue into 4 to 7 mm blocks
Cut tissue into 4 to 7 mm blocks
 Place it from 4 rs to overnig t in cold (+4°C) 3.7% formalde yde solu-
Place it from 4 rs to overnig t in cold (+4°C) 3.7% formalde yde solu-Place it from 4 rs to overnig t in cold (+4°C) 3.7% formalde yde solu-
Place it from 4 rs to overnig t in cold (+4°C) 3.7% formalde yde solu-
tion in PBS
tion in PBStion in PBS
tion in PBS
 Transfer to cold (+4°C) 50% Et anol for 1 r
Transfer to cold (+4°C) 50% Et anol for 1 rTransfer to cold (+4°C) 50% Et anol for 1 r
Transfer to cold (+4°C) 50% Et anol for 1 r
 T en incubate 2 times for at least 2 r eac in cold (+4°C) 70% Et anol
T en incubate 2 times for at least 2 r eac in cold (+4°C) 70% Et anol T en incubate 2 times for at least 2 r eac in cold (+4°C) 70% Et anol
T en incubate 2 times for at least 2 r eac in cold (+4°C) 70% Et anol
(can be left for several days after t is stage)
(can be left for several days after t is stage)(can be left for several days after t is stage)
(can be left for several days after t is stage)
 2 times for 1 r eac in cold (+4°C) 96% Et anol
2 times for 1 r eac in cold (+4°C) 96% Et anol2 times for 1 r eac in cold (+4°C) 96% Et anol
2 times for 1 r eac in cold (+4°C) 96% Et anol
 2 times for 1 r eac in 100% Et anol
2 times for 1 r eac in 100% Et anol2 times for 1 r eac in 100% Et anol
2 times for 1 r eac in 100% Et anol
 1 time for 2 rs in Xylene
1 time for 2 rs in Xylene1 time for 2 rs in Xylene
1 time for 2 rs in Xylene
 2 times 2 rs eac in Paraplast Plus. Do not over eat!
2 times 2 rs eac in Paraplast Plus. Do not over eat!2 times 2 rs eac in Paraplast Plus. Do not over eat!
2 times 2 rs eac in Paraplast Plus. Do not over eat!

7
NOTES
NOTESNOTES
NOTES
 Important.
Important. Important.
Important. Use specialized coated tissue slides for sections t at will
furt er be processed for antigen unmasking. T e best results will be
obtained using Superfrost Plus or Gold or Polylysine-coated Slides
from Menzel-Glazer. Alternatively, you may prepare your own coated
slides (see t e protocol below).
 Important.
Important.Important.
Important. W en t e slides are cut and are dried/spread overnig t,
use t em t e same day. Ot erwise, place slides in a tig tly closed
slide box and store at 2-4°C. T is prevents slow degradation of many
molecules in your sections. According to our experience, storage at
room temperature may dramatically affect immunostaining for some
antigens already wit in 1st week.
CUTTING TISSUE SECTIONS
CUTTING TISSUE SECTIONSCUTTING TISSUE SECTIONS
CUTTING TISSUE SECTIONS
PROTOCOL R2
 All paraffin embedded tissue is cut at a t ickness of 3 µm. T icker
All paraffin embedded tissue is cut at a t ickness of 3 µm. T icker All paraffin embedded tissue is cut at a t ickness of 3 µm. T icker
All paraffin embedded tissue is cut at a t ickness of 3 µm. T icker
sections make morp ological assessment muc more difficult.
sections make morp ological assessment muc more difficult. sections make morp ological assessment muc more difficult.
sections make morp ological assessment muc more difficult.
 T e sections are floated in a warm water bat (45
T e sections are floated in a warm water bat (45T e sections are floated in a warm water bat (45
T e sections are floated in a warm water bat (45
0
00
0
C) before being
C) before being C) before being
C) before being
picked up onto microscope slides and allowed to drain.
picked up onto microscope slides and allowed to drain. picked up onto microscope slides and allowed to drain.
picked up onto microscope slides and allowed to drain.
 Sections for immuno istoc emical staining are picked up on
Sections for immuno istoc emical staining are picked up on Sections for immuno istoc emical staining are picked up on
Sections for immuno istoc emical staining are picked up on coated
coatedcoated
coated
slides and dried overnig t in an incubator at 37
slides and dried overnig t in an incubator at 37slides and dried overnig t in an incubator at 37
slides and dried overnig t in an incubator at 37
0
00
0
C.
C.C.
C.
MAKING APES SLIDES
MAKING APES SLIDESMAKING APES SLIDES
MAKING APES SLIDES
PROTOCOL R3
 Place microscope glasses in slide c ambers, fill c ambers wit 7.5%
Place microscope glasses in slide c ambers, fill c ambers wit 7.5% Place microscope glasses in slide c ambers, fill c ambers wit 7.5%
Place microscope glasses in slide c ambers, fill c ambers wit 7.5%
Rosal Liquid solution in deionized water, leave for 2 rs.
Rosal Liquid solution in deionized water, leave for 2 rs.Rosal Liquid solution in deionized water, leave for 2 rs.
Rosal Liquid solution in deionized water, leave for 2 rs.
 Rinse for 1 r in tap water, t en for 30 min in deionized water
Rinse for 1 r in tap water, t en for 30 min in deionized waterRinse for 1 r in tap water, t en for 30 min in deionized water
Rinse for 1 r in tap water, t en for 30 min in deionized water
 Leave overnig t at 56°C to dry.
Leave overnig t at 56°C to dry.Leave overnig t at 56°C to dry.
Leave overnig t at 56°C to dry.
 Submerge for 5 min in Met anol wit 2% APES (3
Submerge for 5 min in Met anol wit 2% APES (3Submerge for 5 min in Met anol wit 2% APES (3
Submerge for 5 min in Met anol wit 2% APES (3-
--
-
Aminopropyltriet oxysilane
Aminopropyltriet oxysilaneAminopropyltriet oxysilane
Aminopropyltriet oxysilane
,
, ,
,
Sigma A
Sigma ASigma A
Sigma A-
--
-3648).
3648).3648).
3648).
 Rinse for 5 min in Met anol.
Rinse for 5 min in Met anol.Rinse for 5 min in Met anol.
Rinse for 5 min in Met anol.
 Rinse for 5 min in deionized water
Rinse for 5 min in deionized waterRinse for 5 min in deionized water
Rinse for 5 min in deionized water
 Dry overnig t at 37°C.
Dry overnig t at 37°C.Dry overnig t at 37°C.
Dry overnig t at 37°C.
 Incubate for 5 min in deionized water containing 3% Glutaralde yde.
Incubate for 5 min in deionized water containing 3% Glutaralde yde.Incubate for 5 min in deionized water containing 3% Glutaralde yde.
Incubate for 5 min in deionized water containing 3% Glutaralde yde.
 Rinse for 5 min in deionized water
Rinse for 5 min in deionized waterRinse for 5 min in deionized water
Rinse for 5 min in deionized water
 Dry overnig t at 37°C.
Dry overnig t at 37°C.Dry overnig t at 37°C.
Dry overnig t at 37°C.
 Keep t e slides at room temperature.
Keep t e slides at room temperature.Keep t e slides at room temperature.
Keep t e slides at room temperature.
T e APES-solution s ould always be fres ly made. 1L solution is sufficient
for t e treatment of 400 microscope glasses.

8
DE
DEDE
DE-
--
-WAXING SECTIONS
WAXING SECTIONSWAXING SECTIONS
WAXING SECTIONS
PROTOCOL R4
De-wax paraffin sections (t is procedure is identical irrespective of t e
fixative used for t e tissue). Place section(s) into staining vessels, for ex-
ample, Coplin jars, and treat as follows:
 Xylene, t ree c anges, 5 min in eac
Xylene, t ree c anges, 5 min in eacXylene, t ree c anges, 5 min in eac
Xylene, t ree c anges, 5 min in eac
 99% (v/v) Et anol, two c anges, 5 min in eac
99% (v/v) Et anol, two c anges, 5 min in eac99% (v/v) Et anol, two c anges, 5 min in eac
99% (v/v) Et anol, two c anges, 5 min in eac
 Block endogenous peroxidase activity by incubating 30 min in
Block endogenous peroxidase activity by incubating 30 min in Block endogenous peroxidase activity by incubating 30 min in
Block endogenous peroxidase activity by incubating 30 min in
met anol + 0.3% H
met anol + 0.3% Hmet anol + 0.3% H
met anol + 0.3% H
2
22
2
O
OO
O
2
22
2
 90% (v/v) Et anol, one c ange, 5 min
90% (v/v) Et anol, one c ange, 5 min 90% (v/v) Et anol, one c ange, 5 min
90% (v/v) Et anol, one c ange, 5 min
 70% (v/v) Et anol, one c ange, 1
70% (v/v) Et anol, one c ange, 170% (v/v) Et anol, one c ange, 1
70% (v/v) Et anol, one c ange, 1-
--
-3 min
3 min3 min
3 min
 50% (v/v) Et anol, one c ange, 1
50% (v/v) Et anol, one c ange, 150% (v/v) Et anol, one c ange, 1
50% (v/v) Et anol, one c ange, 1-
--
-3 min
3 min3 min
3 min
 30% (v/v) Et anol, one c ange, 1
30% (v/v) Et anol, one c ange, 130% (v/v) Et anol, one c ange, 1
30% (v/v) Et anol, one c ange, 1-
--
-3 min
3 min3 min
3 min
 Deionized water, 2 c anges, 5 min
Deionized water, 2 c anges, 5 minDeionized water, 2 c anges, 5 min
Deionized water, 2 c anges, 5 min

9
Two important issues determine t e c oice of t e buffer:
 T e nature of t e antigen and of t e antibody used for its detection
 T e fixative used and t e degree of fixation.
If your laboratory as good experience wit a routine Citrate (pH6) and
EDTA (pH 8 or 9) buffers you may continue using t ose. We strongly ad-
vise against using wit Retriever Tris-based buffers, including t e “ Uni-
versal” buffers supplied by some companies .
For best results we recommend using concentrated buffers as supplied
by PickCell Laboratories:
R
RR
R-
--
-Buffer A pH 6.0
Buffer A pH 6.0 Buffer A pH 6.0
Buffer A pH 6.0 (in place of Citrate 6)
R
RR
R-
--
-Buffer B pH 8.0
Buffer B pH 8.0Buffer B pH 8.0
Buffer B pH 8.0 (in place of EDTA pH 8 or 9)
R
RR
R-
--
-Buffer C pH 4.5
Buffer C pH 4.5 Buffer C pH 4.5
Buffer C pH 4.5 (very rarely used buffer. Helps to retrieve some antigens)
R
RR
R-
--
-Buffer U pH 6.0
Buffer U pH 6.0Buffer U pH 6.0
Buffer U pH 6.0 (usually t e best buffer to start wit w en you test antibodies
developed against a peptide epitope).
For most routine formalin, formalin-Zn, acid formalin tissues t ese buffers
are optimal wit respect to quality of antigen retrieval and morp ology
preservation.
However, if your routine procedure of fixation is gentle, or you use more
gentle t an routine formalin fixatives, we recommend using t e G variants
G variantsG variants
G variants
(“G” stands for Gentle) of t e same buffers.
T e c oice of t e buffer also depends on t e nature of t e antigen. We ad-
vise you to first run a test for t e most appropriate buffer using t e tissues
w ere t e expression of t e antigen does occur.
General guidelines for c oosing t e buffer:
General guidelines for c oosing t e buffer:General guidelines for c oosing t e buffer:
General guidelines for c oosing t e buffer:
Most of t e nuclear antigens (apoptosis-related, survival-related, prolif-
eration-related, transcription factors) - R-buffer A.
Cell ad esion molecules, cell membrane antigens (extracellular domain)
R-buffer A, rarely buffer B
Cytoskeleton and cytoskeleton-associated molecules - Buffer A
Intracellular domain of some ad esion molecules and surface receptors -
Buffer B or C
Most of antibodies raised against t e linear peptide - Buffer A or buffer U
CHOOSING THE BUFFER
CHOOSING THE BUFFERCHOOSING THE BUFFER
CHOOSING THE BUFFER

10
 Place t e slides into Retriever Slide C amber and immediately fill
Place t e slides into Retriever Slide C amber and immediately fill Place t e slides into Retriever Slide C amber and immediately fill
Place t e slides into Retriever Slide C amber and immediately fill
t em wit t e processing buffer of your c oice ( see t e page 8 for
t em wit t e processing buffer of your c oice ( see t e page 8 for t em wit t e processing buffer of your c oice ( see t e page 8 for
t em wit t e processing buffer of your c oice ( see t e page 8 for
advice on c oosing t e buffer)
advice on c oosing t e buffer)advice on c oosing t e buffer)
advice on c oosing t e buffer)
 Place up to 6 Slide C ambers into t e Rack
Place up to 6 Slide C ambers into t e Rack Place up to 6 Slide C ambers into t e Rack
Place up to 6 Slide C ambers into t e Rack
 Fill t e Body of Retriever wit 750ml of deionized water
Fill t e Body of Retriever wit 750ml of deionized waterFill t e Body of Retriever wit 750ml of deionized water
Fill t e Body of Retriever wit 750ml of deionized water
 Place t e rack wit c ambers inside
Place t e rack wit c ambers insidePlace t e rack wit c ambers inside
Place t e rack wit c ambers inside
 Close t e lid. First, place it in a way t at t e black arrows on bot t e
Close t e lid. First, place it in a way t at t e black arrows on bot t e Close t e lid. First, place it in a way t at t e black arrows on bot t e
Close t e lid. First, place it in a way t at t e black arrows on bot t e
lid and t e body are aligned. Turn t e lid.
lid and t e body are aligned. Turn t e lid.lid and t e body are aligned. Turn t e lid.
lid and t e body are aligned. Turn t e lid.
 Important: control t at t e top valve (
Important: control t at t e top valve (Important: control t at t e top valve (
Important: control t at t e top valve (A
AA
A) is in closed position.
) is in closed position.) is in closed position.
) is in closed position.
 Press t e start button to begin t e processing program.
Press t e start button to begin t e processing program.Press t e start button to begin t e processing program.
Press t e start button to begin t e processing program.
 In approximately 20 min t e program will be completed.
In approximately 20 min t e program will be completed.In approximately 20 min t e program will be completed.
In approximately 20 min t e program will be completed.
 Allow Retriever to cool (takes approximately 2 ours). T is cooling
Allow Retriever to cool (takes approximately 2 ours). T is cooling Allow Retriever to cool (takes approximately 2 ours). T is cooling
Allow Retriever to cool (takes approximately 2 ours). T is cooling
step is a part of t e processing procedure and we advise not to inter-
step is a part of t e processing procedure and we advise not to inter-step is a part of t e processing procedure and we advise not to inter-
step is a part of t e processing procedure and we advise not to inter-
rupt it. T e entire cycle is completed wit in 2.5 ours from t e start.
rupt it. T e entire cycle is completed wit in 2.5 ours from t e start.rupt it. T e entire cycle is completed wit in 2.5 ours from t e start.
rupt it. T e entire cycle is completed wit in 2.5 ours from t e start.
 Open t e pressure valve
Open t e pressure valveOpen t e pressure valve
Open t e pressure valve
 Open t e lid. Take out t e Section Racks and rinse t e slides 3 times
Open t e lid. Take out t e Section Racks and rinse t e slides 3 times Open t e lid. Take out t e Section Racks and rinse t e slides 3 times
Open t e lid. Take out t e Section Racks and rinse t e slides 3 times
wit deionised water or PBS by emptying and refilling t e c ambers.
wit deionised water or PBS by emptying and refilling t e c ambers.wit deionised water or PBS by emptying and refilling t e c ambers.
wit deionised water or PBS by emptying and refilling t e c ambers.
 Leave t e slides in t e last c ange of PBS for at least 15 minutes. We
Leave t e slides in t e last c ange of PBS for at least 15 minutes. We Leave t e slides in t e last c ange of PBS for at least 15 minutes. We
Leave t e slides in t e last c ange of PBS for at least 15 minutes. We
advise proceding t en wit immunostaining. Do not store t e proc-
advise proceding t en wit immunostaining. Do not store t e proc-advise proceding t en wit immunostaining. Do not store t e proc-
advise proceding t en wit immunostaining. Do not store t e proc-
essed sections.
essed sections.essed sections.
essed sections.
PROCESSING SECTIONS
PROCESSING SECTIONSPROCESSING SECTIONS
PROCESSING SECTIONS
PROTOCOL R5
 Important.
Important.Important.
Important. We generally advise you to start t e tissue processing pro-
cedure in t e evening of t e day preceding t e planned immunostain-
ing experiments. For t e best results we advise to allow t e Retriever
to run t e cycle and leave t e sections to cool in buffer overnig t
(over 10 instead of 2 ours).
 For many routine antigens t e processing may be s ortened. In t at
case leave t e sections for 30 minutes after t e processing cycle
stops, t en open t e Retriever by first de-pressurizing t e unit by
opening t e pressure valve (beware of t e steam) and proceed furt er
as described above. However, please c eck on several control slides
for particular antibodies you are using t at t is procedure is suitable
for a particular tissue material and antibodies t at are being used in
your laboratory.