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.

11
The Cycle will run for approxima
tely 20 minutes. The following se
quence of events occurs:
L1 illuminates
L2 illuminates
As the temperature rises, air will be
displaced by steam through the Air
Bleed Device located in the lid.
Pressure Indicator ( ) will rise
in d ica t in g t h e u n it is n o w
pressurized.
The Temperature and Pressure
conditions are reached when:
Lights: L1 illuminates
L2 flashes
L3 illuminates
Cycle is completed
when the Buzzer sounds.
Lights: L1 illuminates
L4 illuminates
(Note: L4 remains until a
new cycle is started or the unit is
disconnected from the mains
power)
GREEN
GREEN
GREEN
GREEN
ORANGE
ORANGE
C
YELLOW
Processing
GREEN
L1
L2 L3 L4 L5
PROCESSING SECTIONS
PROCESSING SECTIONSPROCESSING SECTIONS
PROCESSING SECTIONS
Slide C amber. Up to 18
slides fits one c amber. Place
t e slides in t e same orienta-
tion of t e section side
Place Slide C ambers into t e
Rack and pour t e processing
buffer into t e C ambers to fill
all free space.
Fill t e Body of Retriever wit
750 ml of deionized water and
place t e Rack into Retriever.
Close t e Valve. Pus t e Start button on t e display.
T e lig t indicators (s own on t e left) will be illumi-
nated as t e cycle proceeds.
Close t e lid as s own.

12
 Important: w en c oosing t e detection system for staining proc-
essed sections, aim for a sensitive one. T is s ould ensure a quality
result in t e end.
IMMUNOSTAINING
IMMUNOSTAININGIMMUNOSTAINING
IMMUNOSTAINING
 After processing in Retriever use t e sections wit in t e same day
After processing in Retriever use t e sections wit in t e same dayAfter processing in Retriever use t e sections wit in t e same day
After processing in Retriever use t e sections wit in t e same day
 Rinse t e sections in PBS for 5 min.
Rinse t e sections in PBS for 5 min.Rinse t e sections in PBS for 5 min.
Rinse t e sections in PBS for 5 min.
 Mount t em in a umid c amber of your c oice. At t is stage you may
Mount t em in a umid c amber of your c oice. At t is stage you may Mount t em in a umid c amber of your c oice. At t is stage you may
Mount t em in a umid c amber of your c oice. At t is stage you may
wipe t e drops of liquid around t e section wit a paper tissue and
wipe t e drops of liquid around t e section wit a paper tissue and wipe t e drops of liquid around t e section wit a paper tissue and
wipe t e drops of liquid around t e section wit a paper tissue and
circle t e section wit a special pen to restrict t e area for applying
circle t e section wit a special pen to restrict t e area for applying circle t e section wit a special pen to restrict t e area for applying
circle t e section wit a special pen to restrict t e area for applying
primary and secondary detection reagents.
primary and secondary detection reagents.primary and secondary detection reagents.
primary and secondary detection reagents.
 If necessary (depends on t e quality of t e antibody used) block t e
If necessary (depends on t e quality of t e antibody used) block t e If necessary (depends on t e quality of t e antibody used) block t e
If necessary (depends on t e quality of t e antibody used) block t e
section wit eit er 0.5 % casein solution or wit PickCell’s Block solu-
section wit eit er 0.5 % casein solution or wit PickCell’s Block solu-section wit eit er 0.5 % casein solution or wit PickCell’s Block solu-
section wit eit er 0.5 % casein solution or wit PickCell’s Block solu-
tion.
tion.tion.
tion.
 Leave t e blocking solution on section for 30 minutes
Leave t e blocking solution on section for 30 minutes Leave t e blocking solution on section for 30 minutes
Leave t e blocking solution on section for 30 minutes
 Rinse t e section wit PBS
Rinse t e section wit PBSRinse t e section wit PBS
Rinse t e section wit PBS
 Apply t e primary antibody in an appropriate dilution. In general, we
Apply t e primary antibody in an appropriate dilution. In general, we Apply t e primary antibody in an appropriate dilution. In general, we
Apply t e primary antibody in an appropriate dilution. In general, we
advise an overnig t incubation wit a primary antibody, preferably at
advise an overnig t incubation wit a primary antibody, preferably at advise an overnig t incubation wit a primary antibody, preferably at
advise an overnig t incubation wit a primary antibody, preferably at
4
44
4
o
oo
o
C. T is allows ac ieving a better signal/noise ratio even w en using
C. T is allows ac ieving a better signal/noise ratio even w en using C. T is allows ac ieving a better signal/noise ratio even w en using
C. T is allows ac ieving a better signal/noise ratio even w en using
a not really perfect antibody. Incubation at cold decreases non
a not really perfect antibody. Incubation at cold decreases nona not really perfect antibody. Incubation at cold decreases non
a not really perfect antibody. Incubation at cold decreases non-
--
-
specific binding if suc occurs due to t e properties of t e antibody
specific binding if suc occurs due to t e properties of t e antibody specific binding if suc occurs due to t e properties of t e antibody
specific binding if suc occurs due to t e properties of t e antibody
or of a particular tissue. Incubate your slides in closed umidified
or of a particular tissue. Incubate your slides in closed umidified or of a particular tissue. Incubate your slides in closed umidified
or of a particular tissue. Incubate your slides in closed umidified
c amber, suc as PickCell’s staining tray.
c amber, suc as PickCell’s staining tray.c amber, suc as PickCell’s staining tray.
c amber, suc as PickCell’s staining tray.
 Was t e slides 3 times in PBS 10 min eac .
Was t e slides 3 times in PBS 10 min eac .Was t e slides 3 times in PBS 10 min eac .
Was t e slides 3 times in PBS 10 min eac .
 Apply t e detection system
Apply t e detection system Apply t e detection system
Apply t e detection system
PROTOCOL R6

13
WHAT YOU MUST GET
WHAT YOU MUST GETWHAT YOU MUST GET
WHAT YOU MUST GET
D
E
F
G
EXAMPLES OF PROCESSED TISSUE
A B C
Compare t ese t ree sections of squamous epit elium: Section A
AA
A was
untreated, section B
BB
B was processed in a microwave, and section C
CC
C - in
Retriever. Note t e integrity of t e tissue after Retriever, w ile it is obvi-
ously damaged by microwave.
Here we s ow several examples of immunostaining of formalin fixed, par-
affing-embedded tissue sections processed in Retriever. Standard proc-
essing cycle in buffer A
AA
A (sections E,F,G
E,F,GE,F,G
E,F,G) or buffer B (D
DD
D) was used. T e
antibodies form were used against CD8 (D
DD
D), E-cad erin (E
EE
E), Ki-67 (F
FF
F) and
PCNA (G
GG
G). APES coated slides were used.
T is is a standard quality of morp ology and staining you must expect.
We ave c osen t ese antigens since t ey are widely used as markers
and t e intensity and quality of staining may be evaluated by most of po-
tential users. Please note t e quality of t e tissue morp ology in all
preparations presented.

14
To ensure your Retriever gives you t e years of service for w ic it was
designed, it is important to remember a few "do's" and "don'ts" wit re-
gard to t e operation of t e unit and to carry out t e simple care and
maintenance procedures on a weekly basis.
1 ....you read t ese instructions and always follow t e operating se-
quence.
2.... t e work surface on w ic you will place t e Retriever is flat and
solid.
3.... If you put in racks/staining jars ot er t an t e supplied wit t e Re-
triever, t e material t e former is made of can wit stand t e temperature
and pressure
4.... t e water level is maintained regularly wit clean distilled or de-
ionised water only.
5.... t e unit is in a "draug t free" environment and is positioned not less
t an 250 mm from adjacent walls.
6.... you only use green sealing gasket (219500) and t at it is c anged at
t e end of it's life, if visibly damaged, or w en s rinkage as occurred,
see "Fault mode - 5".
7..... t e lid is securely closed w en t e unit is not in use, to avoid t e risk
of accidental damage.
Never leave in position as s own in F1.
8.... you quote your model details, serial number and date of purc ase
w en contacting PickCell Laboratories or your supplier.
1...touc t e unit w ilst in operation - it gets HOT
2.... attempt to remove t e lid during operation. DO NOT LUBRICATE
GASKET
3.... lose t is operating instruction manual. If lost, please download it at
www.antigenretriever.com
4.... add any c emicals w atsoever to t e water.
5.... attempt to use in buffers volatile substances, toxic materials, or inap-
propriate loads suc as easy melting plastic.
6.... place t e unit on eat sensitive surfaces, polis ed wood or glass.
CONTINUED OPERATION
CONTINUED OPERATIONCONTINUED OPERATION
CONTINUED OPERATION
DO ESURE THAT...
DO OT! ...

15
7.... open t e Depressurization Valve (A) during t e cycle.
8... leave t e Depressurization Valve (A) in t e "open" position w en plac-
ing t e lid upside down on a work surface.
9...immerse t e unit or electrical cord in water w en cleaning.
10...use abrasive materials or lubricants w en cleaning.
11...drop or abuse t e unit.
12...use in areas of risk associated wit flammable materials or
gasses.
13...attempt to c ange fuses until t e unit as been unplugged from
t e mains. Only qualified persons s ould c ange fuses.
14...reac over t e unit w en removing cover, to do so may cause
burns from rising eat and steam.
15...press start button once cycle as been started as t is will reset t e
cycle timer to zero.
CONTINUED OPERATION
CONTINUED OPERATIONCONTINUED OPERATION
CONTINUED OPERATION

16
WARNING! Disconnect t e Retriever from t e main’s power supply be-
WARNING! Disconnect t e Retriever from t e main’s power supply be-WARNING! Disconnect t e Retriever from t e main’s power supply be-
WARNING! Disconnect t e Retriever from t e main’s power supply be-
fore cleaning
fore cleaningfore cleaning
fore cleaning.
Please always use only deinoized water to fill t e internal c amber of
retriever. In t is case no scaling occurs. If some scaling occurs due to
buffer spilled, rinse t e inside of t e Retriever wit lig t de-scaling agent
(like de-scaling liquid for electric cookers and kettles). Rinse wit dis-
tilled water and wipe dry.
If t e unit gets dirty, clean bot interior and exterior wit warm, soapy
water ensuring t e electrical parts are kept dry.
T e sealing gasket in t e lid of Retriever must be cleaned if any leakage
occurs (t e vapor coming from t e side of t e lid, not t roug t e pres-
sure indicator, t e unit as problems wit keeping t e pressure during
t e cycle). To perform t is operation:
1. Remove t e Gasket from inside t e lid and clean wit warm, soapy-
water.
2. Rinse t oroug ly, s ake dry, do not wipe.
3. Replace in t e lid by tucking evenly under all lugs starting at t e Gas-
ket Offset Device. It may appear slig tly wrinkled until used.
4. Replace gasket w en it begins to s ow signs of leakage.
5. If a new gasket leaks, or a persistent leak develops, gently clean t e
sealing surface of bot t e lid and body of t e unit wit a
"Scotc brite" scrubbing pad making sure you do not remove any
metal. Rinse bot surfaces but do not dry.
6. Monitor t e first cycle of t e day to c eck t e Air Bleed device,
w ic is located inside t e lid, audibly "clicks" s ut.
7. Lubricate underside of body lugs wit "Vaseline" if t e lid becomes
stiff.
8. DO NOT LUBRICATE THE GASKET
CARE & MAINTANANCE
CARE & MAINTANANCECARE & MAINTANANCE
CARE & MAINTANANCE
CLEAIG RETRIEVER
GREE SEALIG GASKET

17
TROUBLESHOOTING
TROUBLESHOOTINGTROUBLESHOOTING
TROUBLESHOOTING
Fault 1:
Fault 1:Fault 1:
Fault 1: No
Power to Unit
Lig t: L1 fails
to illuminate
Blown fuse / Defec-
tive Socket / Mains
not connected
Ensure mains lead is connected. C eck /
replace fuse: C eck power to socket:
Fault 2:
Fault 2:Fault 2:
Fault 2: Lig t:
L5 flas es RED
Low water level or
Boil dry.
Allow unit to cool before refilling to t e cor-
rect level. Disconnect from mains t en re-
connect and repeat cycle. If t e fault recurs
wit sufficient water, arrange for a service
engineer to visit.
Fault 3:
Fault 3:Fault 3:
Fault 3: Cycle
failed Lig t: L4
fails to illumi-
nate GREEN
and t ere is no
audible buzzer
Many possible
causes
Disconnect from mains t en reconnect and
repeat cycle. If t e fault recurs arrange for
a service engineer to visit.
Fault 4:
Fault 4:Fault 4:
Fault 4: Steam
or water leaks
from under t e
lid
I) Worn or dirty
gasket.
Was gasket and sealing surfaces on t e
body and lid as described under "Care and
Maintenance. If t e fault persists, replace
wit a new gasket.
ii) Incorrectly
closed lid.
Ensure t e unit is fully depressurized by
opening Depressurization Valve (A). Re-
move lid and refit carefully. Disconnect
from mains, reconnect and repeat cycle
Fault 5:
Fault 5:Fault 5:
Fault 5: Exces-
sive steam or
water leaking
from Depres-
surization
Valve (A).
Depressurization
Valve (A) in
"OPEN" position
Close Depressurization Valve (A).

18
In t e event of a fault occurring during any stage of t e units operation,
identify t e fault by referring to t e descriptions below. T e fault con be
rectified by following t e Fault Remedy applicable to t e problem in-
curred.In t e unlikely event t at somet ing s ould go wrong, we ave in-
we ave in-we ave in-
we ave in-
corporated a number of safety features
corporated a number of safety featurescorporated a number of safety features
corporated a number of safety features to ensure t at your autoclave re-
mains safe at all times.
1.
1.1.
1. Located to t e rear of t e lid, beneat t e cover, is a spring called t e
Gasket Offset Device
Gasket Offset DeviceGasket Offset Device
Gasket Offset Device (GOD Spring), designed to prevent pressure
building up if t e lid as been incorrectly fitted.DO NOT TAMPER
DO NOT TAMPER DO NOT TAMPER
DO NOT TAMPER
WITH THIS SAFETY DEVICE
WITH THIS SAFETY DEVICEWITH THIS SAFETY DEVICE
WITH THIS SAFETY DEVICE
2.
2.2.
2. If for any reason, t e temperature falls below t e minimum required
temperature, resulting in t e Temperature Lig t (L3) switc ing off,
t e cycle timer will re-start.
3.
3.3.
3. If t ere is an electrical or electronic failure resulting in a build up of
pressure - in excess of normal operating pressure - one or all of t e
following safety features will be activated.
i)Depressurization Valve (A
AA
A) will loudly and rapidly "vent" steam.
ii)T e gasket will "extrude" t roug t e slot in t e rear of t e lid
rapidly releasing excess pressure and steam.
iii) A non - resettable t ermal fuse located in t e base of t e unit
will "melt' at a pre-determined temperature, disconnecting t e
power.
S ould any of t e devices listed activate, please observe t e follwing
S ould any of t e devices listed activate, please observe t e follwing S ould any of t e devices listed activate, please observe t e follwing
S ould any of t e devices listed activate, please observe t e follwing
steps:
steps:steps:
steps:
A)
A)A)
A) Do not touc t e unit.
B)
B)B)
B) Switc off at t e wall socket and un-plug
C)
C)C)
C) Allow temperature and pressure to drop before touc ing t e
unit and removing t e content
D)
D)D)
D) Do not attempt to re-start t e unit
E)
E)E)
E) Arrange for an immediate service by contacting PickCell Labs
or your local Distributor.
SAFETY FEATURES
SAFETY FEATURESSAFETY FEATURES
SAFETY FEATURES

19
STANDARDS
STANDARDS STANDARDS
STANDARDS
Prestige Medical, a manufacturer of t e 2100-Retriever ( belongs to t e
2100 Series) as always been proud of its reputation for quality and inno-
vation in t e design and production of our sterilization systems. Prestige
Medical as continually strived to be at t e forefront of t e industry wit
regards to meeting and even exceeding t ose standards w ic apply to it
as a manufacturer and t e products it makes:
ISO 9001 (Certificate No. FM 31988)
ISO 9001 (Certificate No. FM 31988)ISO 9001 (Certificate No. FM 31988)
ISO 9001 (Certificate No. FM 31988) T e international standard for quality. T e
award of t is International Standard confirms our position at t e cutting edge of
customer service and is t e guarantee of continued quality in all areas of product
design, production and servicing.
EN 46001 (Certificate No. 1354)
EN 46001 (Certificate No. 1354)EN 46001 (Certificate No. 1354)
EN 46001 (Certificate No. 1354) T e European quality standard for manufactur-
ers of medical devices. EN 46001 is awarded in conjunction wit ISO 9001 and is
a more stringent set of standards w ic are applicable to manufacturers of medi-
cal devices.
MDD 93/42/EEC
MDD 93/42/EECMDD 93/42/EEC
MDD 93/42/EEC T e European Medical Devices Directive. T is directive, is man-
datory for all products manufactured after t e 14t June 1998, and is t e new
European standard to w ic all medical products must comply. T e directive cov-
ers all aspects of product manufacturing, safety and performance.
EMC 89/366/EEC
EMC 89/366/EECEMC 89/366/EEC
EMC 89/366/EEC T e European standard for Electro-Magnetic Compatibility. T is
directive applies to electrical products t at are tested to ensure t ey do not inter-
fere wit adjacent electrical products, t ey are immune to electromagnetic emis-
sions from ot er electrical products and t ey do not exceed armful levels of
electromagnetic emissions. Products t at meet t is directive can be 'CE' marked.
FDA Good Manufacturing Practice
FDA Good Manufacturing PracticeFDA Good Manufacturing Practice
FDA Good Manufacturing Practice T e award of t is standard (similar to ISO
9001) by t e United States Food and Drug Administration is a quality assurance
system for manufacturing control and is necessary for all companies marketing
medical devices in t e U.S.A. (Product approval is also necessary - via 510(k))
Once awarded, t e FDA conducts re-appraisals every two to t ree years to en-
sure t e standards are being continually ad ered to. Prestige Medical ave sub-
sequently undergone recent (Marc 1999) routine re-assessment to t e new FDA
Q.S.R. (Quality System Requirements), t e latest compliance requirements.
FDA (510k)
FDA (510k)FDA (510k)
FDA (510k) - Series 2100 FDA (510k) for medical devices awarded specifically to
Prestige Medical branded Series 2100, allowing t ese autoclaves to be marketed
in t e U.S.A. T is standard covers all aspects of product performance and effec-
tiveness, wit t e aim of ensuring patient safety at all times.
2100
21002100
2100-
--
-Retriever as been designed to comply in part or fully wit t e fol-
Retriever as been designed to comply in part or fully wit t e fol-Retriever as been designed to comply in part or fully wit t e fol-
Retriever as been designed to comply in part or fully wit t e fol-
lowing standards:
lowing standards: lowing standards:
lowing standards:
IEC601: Electrical and product safety standard.
DIN 58946: Part 8. Type testing for microbiological performance.
EMC: Electro Magnetic Compatibility. EN 50081 - 1, EN 50082 - 1 and prEN 50082
- 2 are generic standards relating to EMC.
BS 3970 Part 4: Britis Standard for autoclaves. Pressure vessel complies.
ESCHLE: Electrical Safety Code for Hospital Laboratory Equipment (UK).
UL 544: Underwriters Laboratory (America) covering electrical and product
safety.
CSA Class 8721 01: Canadian Standards Association covering electrical and
product safety.

20
Heig t ............... 335 mm
Capacity ................. 9 litres
Max. Instrument lengt ........228 mm
Widt ................ 340 mm
Internal c amber
Net Weig t ........ 4.5 kilos
Dimensions (d/ )........210/230 mm
Max. Load Weig t....3.0kg
Fuses
FusesFuses
Fuses - Located under t e control module, fuses F1 OA, 32 x 6.3mm, ce-
ramic sand filled, Mains plug top fuse (User replaceable), F1 3A to
BS1362 UK ONLY.
Rating
RatingRating
Rating - Models are rated continuously for intermittent use.
Body
BodyBody
Body- Deep drawn aluminium.
Lid
LidLid
Lid - Aluminium.
Heater
HeaterHeater
Heater- Externally surface mounted mec anically fixed electric element.
Temperature Cut Out
Temperature Cut OutTemperature Cut Out
Temperature Cut Out - T ermal fuse.
Pressure
PressurePressure
Pressure - Calibrated pressure release valve.
Max. Single Fault Temperature
Max. Single Fault TemperatureMax. Single Fault Temperature
Max. Single Fault Temperature - 133.3°C
Over Voltage Category
Over Voltage CategoryOver Voltage Category
Over Voltage Category - Group II Pollution Degree
Environment Conditions
Environment ConditionsEnvironment Conditions
Environment Conditions - indoor use - temperature 5°C to 40°C - altitude
up to 2000m - maximum relative umidity 80% for temperatures up to 31°
C decreasing linearly to 50% relative umidity at 40°C. - mains supply
voltage fluctuations not to exceed +10% of t e nominal voltage
Input Connections
Input ConnectionsInput Connections
Input Connections - Mains inlet socket ' ot' format conforming to IEC 302.
Safety S ut Down
Safety S ut DownSafety S ut Down
Safety S ut Down - See “Temperature Cut Out”.
Packaging
PackagingPackaging
Packaging - All packaging materials are recyclable.
TECHNICAL SPECIFICATIONS
TECHNICAL SPECIFICATIONSTECHNICAL SPECIFICATIONS
TECHNICAL SPECIFICATIONS
TECHICAL SPECIFICATIOS OF RETRIEVER 2001
OTHER SPECIFICATIOS OF RETRIEVER 2001