IHC_PRINCIPLES.pdf

Basic
SPECIMEN
NM-123
Colon
carcinoma
123456789

Source:
• Carson F., Hladik C. HISTOTECHNOLOGY 3rd Edition
• Diagnostic Immunohistochemistry NORDICQC Krakow,
Poland, 12th-13th October 2015 (seminars)
• Dako’s Guidebook to Immunohistochemical Staining
Methods

In the clinical laboratory:
 a heavily used technique,
 for assisting the pathologist in making diagnosis,
Used to determine:
 the origins,
 prognosis,
 and treatment of a tumor
Rigorous quality control (QC)
 Accurate and consistent results
 Expected staining patterns
Trobbleshooting:
 Fixation
 Processing
 Tissue types

Grossing
Tissue processing
Embedding
Sectioning
Staining
HE/HC/IHC
Tissue sections
fixation
fixation
Diagnosis

• Fixation & tissue preparation
• Pre-treatment/epitope retrieval
(Appropriate and efficient epitope
retrieval)
• Primary antibody selection
(Appropriate choice & titre of
antibody/clone)
• Detection system s
pecific & sensitive detection system
• Appropriate choice of
control material
Important considerations
for IHC

START
IHC Images by Kornstein, MD, Medical College of Virginia
Abnormal 2+ Abnormal 3+
Normal 0
Normal
Normal 1+
Normal Abnormal low
amplification
Abnormal high
amplification
ALMOUST
THE END
Important considerations for IHC

Tissue preparation standards
1. PRELAB PITFALLS – 68%
2. FIXATION TIME: 6 - 48 h – PRECICION???
3. FORMALIN TEMPERATURE....
ROOM TEMPERATURE...WINTER//SUMMER???
4. FORMALIN pH !!! 7.0
5. FORMALIN VORTEXING
6. FRESH FORMALIN???...

LITTLE BIT OF HISTORY…
 The term “antibody” was coined by Paul Ehrlich in 1891.
 The principle of IHC has been known since the 1930s,
 It was not until 1942 that the first IHC study was reported…(Coons et al. used FITC-labeled antibodies to identify Pneumococcal
antigens in infected tissue).
 1974 Taylor and Burns developed IHC on routinely processed FFPE tissues
 1975 Köhler and Milstein presented the hybridoma technique to produce monoclonal antibodies (mAbs) by fusing an antibody-
producing B cell with a myeloma cell that is selected for its ability to grow in tissue culture
 1979 Peroxidase anti-peroxidase technique
 early 1980’s avidin & biotin complex
Since then, improvements have been made in protein conjugation, tissue fixation methods, detection labels and microscopy,
making immunohistochemistry a routine and essential tool in diagnostic and research laboratories

retrieval)
antibody/clone)
• Detection system s
pecific & sensitive detection system
• Appropriate choice of control
material
for IHC

Fixation & tissue preparation
Fixation is a complex series of chemical events that differ for the
different groups of substance found in tissues.
• A fixative is described as a chemical substance which will
preserve the shape, structure, relationship and chemical
constituents of tissues and cells after death.
The aim of fixation according to IHC:
1- during fixation, hydrogen bonds are formed – which must be reserved to have successful
antibody binding to the antigen site.

– preservation of tissues in its original condition.
Aldehyde based
• 10% NBF
• 4% formaldehyde with PBS buffer
• 2% formaldehyde with picric acid and PBS
• Immersion v. transcardial perfusion

16
- factors affecting fixation good quality of IHC
• Formalin is best at 10% pH 7.2 (7.0 -7.4)
• Too high a concentration may adversely affect the
tissues and produce artefact similar to excessive
heat.
40% formaldehyde 250 ml
- Na2HPO4 x H2O 6,5 g
- NaH2PO4 4,0 g
- H2O dest. 750 ml

General principles of fixation
 Amount of fixing fluid should be approx. 20 times more than the volume of
tissue held in a container with a required fixation time.
Temperature has an important effect. ROOM TEMPARATURE: 21°C
A lower temperature retard fixation – reduce autolytic reaction.
 A higher temperature will decrease the required for fixation but will
increase autolysis.
• Formalin is best at 10% pH 7.2 (7.0 -7.4)

FIXATIVES FOR PARAFFIN-PROCESSED TISSUE
- new regulation regarding predictive marker staining  responsibility on the
clinical laboratory fo dokumenting the time of fixation
- Time: 6 - 48 h – standard: 24 h
The speed of fixation of most fixative is almost 1 mm/hour
(according to Medawar penetration index: 1 mm/ 1 godz.
d = k √ t (d = depth k = penetration index t = time (h)

Penetration time at K = 3.6( d = K x √t )
• 1 hour = 3.6 mm
• 4 hours = 7.2 mm (1.8 mm/hr)
• 16 hours = 14.4 mm (0.9 mm/hr)
• 64 hours = 28.8 mm (0.45 mm/hr)
• 256 hours = 57.6 mm (0.225 mm/hr)
(to double the depth takes 4x the time)
NORDICQC, seminars 20

100 % binding of formaldehyde after 24 hours at 25°C
50 % binding of formaldehyde after 100 min. at 25°C room temp.37˚C
Helander, KG. Kinetic studies of formaldehyde binding in tissue.
Biotechnique and Histochemistry. 1994; 69, 177 -179 NORDICQC, seminars 20

Am J Surg Pathol, Vol. 24, No. 7, 2000
Reaction!!!!!
− Penetration is just a part of the problem
− The main clue !!!!!  reaction!!!
− „GOLDEN HOUR” – first hour
is the most important during fixation
we can save or completly destroy the tissue
(IMMUNOREACTIVITY)

Appropriate tissue fixation and processing
• – Problem 1: Delayed fixation
• – Problem 2: Too short fixation in NBF
• – Problem 3: Other fixatives than NBF
• – Too long fixation in NBF is not a problem !!!??
Appropriate tissue fixation and processing
NORDICQC, seminars 20

• Because fixation varies markely from laboratory to laboratory
• Vimentin provides an excellent way of determinating when tissue has been
overfixed.
• Vimentin staining is usually excelent in paraffin sections of tissues that have
been optimally fixed in formalin but is progressively lost as the lenght of
time in the fixative increases.
• When the vimentin staining is completely negative  the tissues are
overfixed and all antibodies should be interpreted with caution.
• When the preservation of vimentin is uneven, then all immunostains should
be read in the area of most intense vimentin staining
DAKO, seminars 2015

Tissue preparation standards NEW ALTERNATIVE

TissueSafe – urządzenie, w którym tkanki możemy
opakować w folię, w próżni co eliminuje konieczność
utrwalenia jej w miejscu pobrania (np. blok
operacyjny).
- Urządzenie to pozwala również na transport
tkanek z miejsca pobrania do laboratorium
histopatologicznego.
- Tkanki przewożone są w temperaturze 4 °C.
- Otrzymaną tkankę utrwalamy dopiero po wyjęciu z
worka próżniowego, w laboratorium.
- Od początku możemy nadzorować proces
utrwalania materiału.

Ventana: System 2 + 2
1. Fresh tissue material – on ice – department of clinical pathomorphology
2. Cutting the fresh material
3. Placing the fresh thin tissue sections into 10% buffer formalin for 2 h in 4°c
4. Last step of fixation: 2 h in 45°c
Chafin D, Theiss A, Roberts E, Borlee G, Otter M, et al. (2013) Rapid Two-Temperature Formalin Fixation.
PLoS ONE 8(1): e54138. doi:10.1371/journal.pone.0054138

Tissue preparation standards

Estrogen receptor expression at
different delayed formalin fixation
times (0 minutes, 4 and 8 hours,
and overnight).
Note the decreased
number/percentage of positive cells
and the intensity of the stain with
increased time of delayed fixation.

Sectioning
• Paraffin
• Most commonly used
• Must heat and process through xylenes and alcohols – ruins some
antigens
• BEST if not stored more than two weeks – lose antigenicity after
that time
Sections usually 3-4µm
HER2 IHC = 4µm
Thinner sections 2-3µm required for:-
Bone marrow trephines
Lymph nodes
Renal biopsies
Thicker sections 6-20µm required for:-
Cases for amyloid (histochemistry)
(6-8µm)

Model A22
Zalecane do bardzo
cienkich sekcji w
krojeniu rutynowym i
badaniach
naukowych. Nadaje
się do tkanki
włóknistej, a także do
cięcia
wstążeczkowego.
Długość - 80mm;
Wysokość - 8 mm; Kąt
żyletki - 22°;
Model A35
Zalecane do bardzo
cienkich sekcji w
krojeniu rutynowym i
badaniach
naukowych. Nadaje
się zarówno do
tkanki miękkiej jak i
twardej .
Długość - 80mm;
Wysokość - 8 mm;
Kąt żyletki - 35°
Model C35
Zalecane wyłącznie
do skrawania w
kriostatach w celu
uzyskania bardzo
cienkich sekcji.
Jedyna żyletka
wykonana ze stali
węglowej.
Długość - 80mm;
żyletki - 35°
Model R35
Zalecane do
skrawania
wstążeczkowego.
Nadaje się przede
wszystkim do tkanki
twardej, możliwe jest
jednak również
skrawanie materiału
miękkiego.
Długość - 80mm;
żyletki - 35°

Model N35
Zalecane do
skrawania bardzo
twardych materiałów
w krojeniu
rutynowym.
Długość - 80mm;
Wysokość - 8 mm;
Model N35HR
Zalecane do
skrawania bardzo
twardych i trudnych
materiałów zarówno
w skrawaniu
rutynowym jak i
wstążeczkowym.
Długość - 80mm;
Wysokość - 8 mm;
Model S35L
Zalecane do
skrawania
rutynowego tkanek o
dużych wymiarach
zatopionych
w bloczku
parafinowym oraz
większych biopsji.
Długość - 120 mm;
Wysokość - 8 mm;
Model S35LL
Zalecane do
skrawania
rutynowego tkanek o
bardzo dużych
wymiarach
zatopionych
w bloczku
parafinowym.
Długość - 180 mm;
Wysokość - 8 mm;

Tissue preparation - sectioning

H&E
Score lines visible on the
waterbath
David Muskett - SD/CP5 Microtomy

This short ribbon of sections that was cut from a cold block shows considerable compression
(30–40%). In this case re-setting the knife tilt angle overcame the problem.
Optimise knife tilt angle

This block face has cracked because it was frozen to –15 °C in a freezer prior to cutting. The
cracks may make sectioning and flotation difficult because the wax is no longer bound to the
tissue. David Muskett - SD/CP5 Microtomy
Avoid freeze damaging

Carefully choose a section

Pre-treatment/epitope retrieval
(Appropriate and efficient epitope retrieval)
two common categories of retrieval methods
1. HIER – HEAT-INDUCED EPITOPE RETRIEVAL
2. EIER – ENZYME-INDUCED EPITOPE ETRIEVAL
• Those methods are necessary to break down the hydrogen bonds,
formed during fixation
• Overfixation  results in in loss of immuneactivity  false negative
results
for IHC

Advantages in epitope retrieval:
- Ability to furthere dilute antibodies
- Exposure of epitopes sites not previously detectable
- More intense reactions with decreased incubation times
- More uniform staining
- Decreased background staining
- Possibility of better standarization
Pre-treatment/epitope retrieval
(Appropriate and efficient epitope retrieval)

Antigen retrieval Pre-treatment / Epitope retrieval:
Defined as an unmasking method
for ”re-storing” blocked antigens
in formaldehyde fixed tissue
The key to an optimal IHC reaction…

HIER Heat Induced Epitope Retrieval
Citrate 6.0//Tris-EDTA 9.0//EDTA 8.0 / 9.0
95°C 20 min.

IHC Procedure validation
Antibody dilution 1:25 1:50 1:100
Antigen retrieval 1. HIER low pH
2. HIER high pH
3. Enzyme
(protease)
1.HIER low pH
2.HIER high pH
3.Enzyme
(protease)
1.HIER low pH
2.HIER high pH
3. Enzyme
(protease)
Incubation 1. 30 min. 37°C
2. 1 h RT
3. 16 h 4°C
1. 30 min. 37°C
2. 1 h RT
3. 16 h 4°C
1. 30 min. 37°C
2. 1 h RT
3. 16 h 4°C

retrieval)
antibody/clone)
• Detection system specific &
sensitive detection system
• Appropriate choice of control
material
for IHC

Monoclonal v. polyclonal
Antibodies monoclonals
• Mouse monoclonals - one clone
to one antigen determinant
• High specificity - low to high
sensitivity
• Mouse or rabbit hybridoma
Primary antibody selection

Rabbit polyclonals - more clones to more antigen determinants
• Low to high specificity - high sensitivity
• Polyclonal antibodies
• Many different species
• Tends to have more non-specific reactivity
• Can have very different
• avidity/affinity batch-to-batch
• Rabbit monoclonals -
• High specificity – high sensitivity
 Polyclonal antibodies reacting with various epitopes
 Each antibody is made by a different B-cell
Monoclonal v. polyclonal

Antibody specifiacation sheet:
• The antibody specification sheet provided with a commercial antibody contains
valuable information regarding the antibody.
• The expiration date under recommended storage conditions
• Long-term//short-term storage temparatures (some antibodies require long-term
storage at -20°C. Measures to ensure that the antibody storage condition is optimal
will assist with antibody stability and maximum reactivity for the life of the antibody.
• The protein concentration and suggested working dilution ranges
• Pretreatment (antigen retrieval) solution; pH, and//or method if recommended
• Expected positive and negative tissue types INCLUDING NORMAL AND TUMOR
• Reference and publication list, and applications of antobody
• ANTIBODY TYPE: IN VITRO (IVD), RESEARCH USE ONLY (RUO), FOR RUTINE
DIAGNOSIS

Prediluted and concentrated antibodies
• Commercialy available – READY TO USE antibodies –
validated
• Concentrated antibodies – dilutions must be performed, to
find opitimal dilution for antigen detection
CONTROL REACTIONS – ANTIBODY VALIDATION

CONTROL REACTIONS – ANTIBODY VALIDATION
• EXAMPLE OF STANDARD LABORATORY PROTOCOL:
• Antigen retrieval - combination of heating method/
• - combination of time
• Peroxidase quenching – peroxidase block
• – combination of time
• Non-specific binding block – combination of time
• Detection system - universal polymer//HRP//AP
• Staining times: - primary antibody combination
• 30 minute//1 hour//16 hours
• - detection system//chromogen
• 20 minutes //1-10 minutes

v

Validated antibody
• IF v. IHC with fluorescence
• WB, ELISA, IP, etc.
Whole molecule or specific portion of epitope?

1:800
1:400
1:50 1:100 1:200
IHC staining results of serially diluted Chromogranin A
antibody on pancreas.
Primary antibody selection rules

At 1:50, the Islet cells stain strongly but there is also a
strong background staining.
1:50
background staining
Islet cells

At 1:800, there is no background staining but the Islet
cells stain very weak.
1:800

At 1:200, there is good contrast and no background
staining, it is therefore the optimal working dilution.
1:200

Primary Antibody Incubation Time / Temparature
• Incubation time is inversely proportional to antibody concentration.
• Higher concentration of antibody allows shorter incubation time.
• 30 minutes 37°C // 1 hour RT // 16 hours 4°C
• Humidity chambers must be used when incubating at higher
temperature to prevent drying of tissue sections.
• 16 /4°C – allow sometines to better antibody penetration – used in
VALIDATION

IHC rules: Blocking
Background staining
• Specific
• Polyclonal antibodies – impure antigen used
• Inadequate fixation – diffusion of antigen – often worse in
center of large block
• Non-specific
• Endogenous enzymes peroxidases//AP

Structure of DAB
NH3
+
NH3
+
+H3
N
+H3
N 4Cl
-
DAB stained slides can be coverslipped with permanent organic
based mounting media.
In some IHC procedures, the dark brown reaction product can
be modified and intensified by adding metals (copper or
cobalt) to DAB solution.
Commonly used enzyme labels for IHC procedures include
horseradish peroxidase (HRP) HRP, from horseradish plant, is an enzyme that
catalyze the reduction of hydrogen peroxide (H2O2) to water and oxygen.
alkaline phophatase (AP)
Commonly used chromogens for HRP include
3-amino-9-ethylcarbazole (AEC)
3,3’-diaminobenzidine (DAB)

• Horse Radish Peroxidase (HRP)
• + high sensitivity
• + precise chromogenic reaction
• + can be amplified by metal (Cu/Ni)
•
• Alkaline Phosphatase (AP)
• + suitable for cryostate sections and cytology
(hematology)
• + double IHC staining
• - granular chromogenic reaction
• - relative low sensitivity
Enzyme

Control material can be categorized in different ways:
„
„
Reagent controls
– Positive reagent control is the actual reagent;
e.g. the primary antibody when tested on control material.
Testing is done during development and product validation to
ensure reagent specificity and sensitivity.
– Negative reagent control is a reagent substitute for the
primary antibody used to assess potential specificity
issues/false positive staining reaction
„
„

Tissue controls
– Positive tissue control is tissue with the specific antigen
at known, relevant and stable level.
Purpose is to docu-ment correct staining.

Negative tissue control
-is tissue without the specific antigen present – or not present in
specific regions.
- Purpose is to document specificity of the staining.
Tissue blocks/tissue microarrays,
- consisting of a few to several different
tissues that may serve as control material
for a range of antibodies

Internal tissue controls
– The presence of the target antigen (protein) within normal
elements of the tissue under investigation is an internal positive
control
„
„

„
„
Cell line controls
– Control material based on cultured cells with specific
antigenic characteristics. This type of control typically is made
for very specific purposes, in particular predictive assays (HER2)

Controls in Daily Routine Testing
On-slide positive tissue controls,
where specific controls are
placed on the same slide as the test
specimen are strongly preferred.
A) Illustration of the principle of
multi-core blocks. Tissue controls are
the cores in red, yellow, gray and
green. Patient sample is placed below
B) Example of on-slide tissue
controls. TMA
C) Example of cell line controls.

Tissue Controls
- both positive and negative tissue controls provide important information
and must be included in daily routine regardless of type of detection
system used.
- the tissues used for controls are carefully selected from normal or
cancer tissues previously analyzed in the laboratory.
- However, in some cases the actual patient tissue being tested can be
used as tissue control. HER2 - gastic
- For all tissue controls, if the staining does not perform as expected,
results from the respective test specimen should be considered invalid.

Positive tissue control
- The positive tissue control must contain the target
antigen at relevant, known and stable expression
level.
- It serves to document that proper staining has been
performed and confirms that the target retrieval procedure
has been carried out correctly.
- Thus, a positive tissue control assesses correct staining
protocol performance (temperature, time and correct
application of reagents).

Positive tissue controls
- are indicative of properly prepared tissue, they should
be as accurate as possible in the same manner as
the patient samples.
- Optimally, autopsy/biopsy/surgical specimens should be
fixed, processed and embedded as soon as
possible for best preservation of antigens.
- Generally, autopsy tissue is least preferred because of
inevitable delays before fixation (degradation of some
antigens)

Control material
On-slide positive tissue controls
-In the daily routine, positive tissue controls may be run on a
separate slide - as a batch control or daily control, or they
may be included on the same slides as the test specimens.
- on-slide positive controls, are
specific controls are placed
on the same slide as the test
specimen strongly preferred

Control material
More and more laboratories are using TMA cores as
on-slide controls.
-building a a few basic multi-tissue control blocks
- each containing a small number of control tissues to
cover most of the markers used in clinical
diagnostics.

Control material
The multi-tissue control blocks contains:
appendix, tonsil, pancreas and liver,
The reaction patterns are described for approximately 100 different
antibodies.

During immunohistochemistry:
Be focus on technical
calibration and controls for validation and verification.

Etapy reakcji IHC Odczynniki Warunki: Czas /Temperatura
Deparafinizacja
Bufor cytrynianowy – pH low
Bufor EDTA – high pH
Uwodnienie
Odkrywanie antygenów 20 min PT-link(95 - 99 °C)
Płukanie
Blokowanie endogennej peroxydazy
Płukanie
Woda redestyl//PBS
H2O2
PBS
2x // 10min
15 min RT wytrząsarka
10 min.
Blokowanie miejsc nieswoiście
wiążących przeciwciała
Roztwór BSA 5% 10 min, RT, komora wilgotna
Inkubacja z pierwszorzędowym
przeciwciałem
Płukanie
Rozcieńczenia:
1:250
PBS
16 godz., temp 4 °C, komora
wilgotna
10 min.
Inkubacja z drugorzedowym
przeciwciałem
płukanie
EnVision Anti-Mouse/Rabbit
FLEX HRP DAKO
PBS
20 min, 37 °C, komora wilgotna
10 min.
Inkubacja z substratem dla
peroksydazy
DAB Chromogen 2 min, RT, komora wilgotna

Step by Step IHC Staining Method
Remove paraffin wax and hydrate
tissue section.
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
Deparaffinization and Rehydration
paraffin wax coated slide

Remove paraffin wax and hydrate
tissue section.
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
tissue section

20:00
Wather bath for 20 minutes using
Antigen Retrieval Solution pH 9.0.
Rinse
Antigen Retrieval
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA

SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
3% hydrogen peroxide block solution for 5 minutes to
inactivate endogenous peroxidase activity.
Block Endogenous Peroxidase
Rinse
Antigen Retrieval
Rinse

Thorough rinse in distilled water and
wash 2 times in PBS buffer.
Rinse
Antigen Retrieval
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA

5% bovine serum albumin (BSA) to block nonspecific
staining.
Protein Block
Antigen Retrieval
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA

Optimally diluted primary antibody for 20 minutes.
Primary Antibody
Antigen Retrieval
Protein Block
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA

Wash 2 times in PBS buffer.
Antigen Retrieval
Protein Block
Primary Antibody
Rinse
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA

Secondary antibody for 20 minutes.
Secondary Antibody
Rinse
Antigen Retrieval
Protein Block
Primary Antibody
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA

Wash 2 times in PBS buffer.
Antigen Retrieval
Protein Block
Primary Antibody
Secondary Antibody
Rinse
Rinse
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA

DAB chromogen for 1 minute.
DAB Chromogen
Antigen Retrieval
Protein Block
Primary Antibody
Secondary Antibody
Rinse
Rinse
Rinse
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA

SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
rinse in water.
DAB Chromogen
Antigen Retrieval
Protein Block
Primary Antibody
Secondary Antibody
Rinse
Rinse
Rinse
Rinse
Rinse

Counterstain in Mayer’s Hematoxylin
for 1 minute.
Antigen Retrieval
Protein Block
Primary Antibody
Secondary Antibody
DAB Chromogen
Rinse
Rinse
Rinse
Rinse
Rinse
Hematoxylin Counterstain
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA

SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
Thorough wash in tap water to “blue” the nuclei.
Antigen Retrieval
Protein Block
Primary Antibody
Secondary Antibody
DABChromogen
Rinse
Rinse
Rinse
Rinse
Rinse
Rinse

Coverslip using mounting medium.
Antigen Retrieval
Protein Block
Primary Antibody
Secondary Antibody
DAB Chromogen
Rinse
Rinse
Rinse
Rinse
Rinse
Rinse
Mount
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA

“Stronty formalinowe", albo
"hematyna kwaśnej formaliny".
Pigment ten tworzy się w środowisku
kwaśnej formaliny.
Hematyna powstaje z hemoglobiny,
reagującej z formaliną w środowisku
kwaśnym, stąd największe
nagromadzenie tego barwnika
obserwowane jest w preparatach
zawierających dużo krwinek.

Utrwalanie materiału tkankowego

Results of incomplete fixation

Improving antibody penetration
 Need this for intracellular (cytoplasmic, nuclear) or
membrane components when epitope is inside cell
membrane
 Detergents most popular
 Triton-X
 Tween
 Also decreases surface tension – better coverage
 Can’t use for membrane proteins
 Acetone/Methanol
 Precipitate proteins outside cell membranes- more accessible
 Saponin
 Punches holes in cell membrane – holes close up when
removed

IHC_PRINCIPLES.pdf

Recommended

Recommended

More Related Content

Similar to IHC_PRINCIPLES.pdf

Similar to IHC_PRINCIPLES.pdf (20)

More from Dinu85

More from Dinu85 (15)

Recently uploaded

Recently uploaded (20)

IHC_PRINCIPLES.pdf