Transcript IHC Methords

IHC Submission
All IHC slides submitted to the core
should be accurately described on
both form and slides. We can
perform the experiment without
rechecking them.
IHC Method
Indirect Alexa 488 or 568
Biotin-Streptavidin system
Indirect Alexa 488 or 568
• A perfect spectral match for FITC filters
• Brighter conjugate fluorescence
requires less antibody and still get
optimal results.
• Superior photostability—allows more
time for image observation and capture.
Biotin-Avidin/Streptavidin System
Biotin-Streptavidin system
• The Biotin-Avidin/Streptavidin System can
improve sensitivity because of potential
amplification with multiple site binding.
• Only a single labeled conjugate, avidin or
streptavidin, need be kept on hand since it
can be used with a variety of biotinylated
lectins, antibodies or probes.
Indirect Alexa 488 or 568
Biotin-Streptavidin system
• High antigen expression tissue
• Inexpensive antibody
• Used when ImmPRESS causes strong
background
• Biotin-Streptavidin is used for:
Goat red fluorescence
Blue fluorescence (AMCA Streptavidin)
Avidin/Biotin Complex (ABC)
Avidin/Biotin Complex (ABC)
• ABC systems are extremely
sensitive
• Can be used to detect any
molecule that is biotinylated
• We use it when ImmPress is not
available
ImmPRESS Polymer
ImmPRESS Polymer Detection
The ImmPRESS™ staining system
- Highly sensitive
- Ready-to-use
- One-step, non-biotin detection
system
- Low background
ImmPRESS Polymer Detection
•
•
•
•
DAB (Diaminobenzidine), brown
Vector VIP, purple
Vector SG, blue-gray
AEC (3-amino-9-ethyl carbazole)*, red
Tyramide Signal Amplification
(TSA™)
Tyramide Signal Amplification
(TSA™)
TSA significantly improves sensitivity
of Immunohistochemistry, (IHC), or
in situ hybridization (ISH).
Conserve precious antibodies or
probes
Tyramide Signal Amplification
(TSA™)
• Tissue with low antigen expression
• Expensive antibody or only small
volume of antibody available
• High-specific antibody
• Low-background tissue sections
Pretreatment
1. Blocking nonspecific binding site
Use Normal serum from the secondary
host to block the nonspecific binding site
2. Block endogenous enzyme interference
Use H2O2 to prevent endogenous
peroxidase
Pretreatment
1. Heat Antigen Retrieval:
Using a Pressure Cooker and different buffer
Bull’s Eye decloaker pH 6.0 (PCBE)
EDTA decloaker pH 8.5 (EDTA)
Borg decloaker pH9.5 (Borg)
Heat treat in pressure cooker at 120oC for
30 seconds, Cool down to 84-86oC.
Pretreatment
2. Protease Antigen Retrieval:
Using Proteinase K, Trypsin or
Pepsin
Incubate time depend on the tissue
10-15 minutes
Label IHC slides
• Use pencil to label the slides
• write the Ab information on top middle
of the slide. Keep the edges free of
writing.
• labeling information for each single or
double staining is shown on your
personal IHC file
Label IHC slides
• Primary Antibody Abbreviation
• Primary Antibody Dilution
• Detection Method (add +H if you want nuclear
counter-stain)
• Date (the Monday of the week of submission
– this must correspond to the date on the
submission form)
• Name Initial
Label IHC slides
YAP R
1:50
ImmPF+H
3/12
KE
Label IHC slides
2H3
3/12
YAP R
1:50
ImmPF+H
KE
Label IHC slides
1 heat
2 heat+H2O2
N no heat
P Pk
T Trypsin
E EDTA
B Borg
G
H
R
N no blocking
1
2
3
4
5
6
7
8
9
488
568
ImmPF
ImmP/F
ImmP/Red
StreF
StreRed
ImmPRed
ImmPHRP
How to fill out IHC form
1. Import the files from template list
to your personal IHC form
2. Duplicate the files from your
personal IHC form
3. Submit a new antibody to
Histology Core for testing.
Pick up right slides for IHC
1. Pick one slide from each sample for IHC
2. Look your slides under microscope to
find your interested area and pick right
slide from each sample for IHC
IHC single staining
Duplicate from your personal IHC form
or import from the IHC template list
IHC Double staining
1. Duplicate from your personal IHC form
or import from the IHC double template
List
2. Duplicate first primary Ab from your IHC
form or import first primary Ab from
Template List. Manually add second
primary Ab to the form
Which Ab to use as first
primary Ab
1.
2.
3.
4.
Primary Ab from goat
GFP, RFP
Strong IHC staining
No treatment
IHC Submission
IHC submission deadline is Thursday.
Generally you will get the IHC slides one
or two weeks.
Fluorescent stained slides will be delivered
to a slide box in each Lab -20oC freezer.
HRP stained slides will be delivered on
Friday to the shelf on Histo Core supply
table.
Histology Submission
1. Send all histology submission to Lan
and MinMin
2. Write correct email subject on your email
( Tissue form, IHC form, InSitu form)
Other questions should be specified
3. Write priority on email subject and copy
email to your PI If your histology request
is urgent