Sample protocol for comparing ANTIBODY DETECTION METHODS
Direct secondary, tyramide amplified, enhanced amplification (TSA-Plus)
Background:
There are multiple methods of detecting antibodies, chromogenic, direct-fluorescent, and the
highly sensitive Tyramide signal amplification in both the standard and enhanced (Plus)
formats. Following is a step by step comparison method protocol.
MATERIALS and REAGENTS:
CitriSolv
2-propanol
Hydrogen peroxide 30% (H2O2) solution
PBS, pH7.2 – 7.4
0.01 M Citrate Buffer pH 6.0
PBS-Blocking Buffer containing 1% BSA,
0.3% Triton and 0.2% powdered non-fat
skim milk
50% Glycerol PBS solution
TM
DMSO (to reconstitute the TSA
reagents)
TM
TSA Fluorescein System (PerkinElmer)
NEL741001KT
•
•
•
Primary antibody
Secondary antibody
Anti-fluorescein antibody (PerkinElmer)
NEF710
nail polish, single edge razor blade
cover glasses, absorbent paper towels
PAP barrier pen (RPI, Sigma, Vector)
Humidity chambers (e.g. plastic slide
folders)
TSA TM-Plus Fluorescein System
(PerkinElmer) NEL701A001KT
Tissues: normal adult mouse brain tissue one section fixed overnight in 4OC Bouins,
one in 4% para-formaldehyde
Five m thick sections were saggitaly cut from fixed paraffin-embedded tissues and
mounted on SurgiPath Snowcoat X-tra™ glass slides. Slides were baked at 60oC for 60
min to ensure adherence to the glass.
Antibody Information
ANTIGEN
Glial Fibrillary
Acidic
Protein(GFAP)
Donkey antiRabbit
Fluorescein
Sheep antiFluoresceinHorse Radish
Peroxidase
Product
Species
Dilution
COMPANY
Z0334
Rabbit
polyclonal
1:50,000
1:5,000
Dako
Cytomation
711015152
Donkey
1:200
Jackson
Immuno
Research
www.JacksonImmuno.com
1:200
PerkinElmer
Life
Sciences
www.las.perkinelmer.com
NEF710
Sheep
Website
www.dakousa.com
DakoCytomation: Rabbit anti-GFAP polyclonal Ig lot;00019620
Z0334
PerkinElmer Life Sciences, Inc: Sheep anti-fluorescein-HRP lot;203469 NEF710
Jackson ImmunoResearch Labs: AffiniPure Donkey a-Rabbit (IgG)-fitc 711-015-152
TSA™ Plus Fluorescein System, for 50-150 Slides: NEL741001KT
TSA™, Fluorescein System, for 50-150 Slides: NEL701A001KT
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PROCEDURE:
We have found good deparaffinization and rehydration of tissue samples can be achieved with
xylene substitutes that contain d-limonene, a naturally occurring citrus compound which is less
toxic than xylenes (e.g.: CitriSolv, HemoDe etc.). After soaking in two changes of CitriSolv (5
and 10 minutes) the slides are lowered into 3 successive baths of 2-propanol (5 minutes each).
Washing and rehydration are completed by 2-3 minutes in gently running water followed by
dH2O for 5 minutes. It is easiest to label the slides before deparaffinization.
For many antibodies antigen detection is enhanced by a heat activated epitope retrieval
process (Antigen Retrieval) in various solutions. 0.01M Citrate Buffer pH 6.0 is our routine
solution due to its low cost and effectiveness. Other solutions may vary pH, detergents or
buffering compounds. Place the slides in a generous amount of buffer in a covered TPX plastic
slide container (VWR 25460-907 or similar) with sufficient water in the steamer to last 30
minutes (about 150 ml for our Aroma rice steamer). Gently steam 20 minutes (after the
solution has come to steaming) in the steamer followed by a minimum 30 minutes of gradual
cooling down to room temperature. Slides are washed in gently running water followed by
dH2O and PBS for 5 minutes each.
Tyramide Signal Amplification is catalyzed by Horseradish Peroxidase, therefore endogenous
peroxidases need to be quenched by exposure to 3% H2O2 in PBS for 10 minutes. Rinse in
dH2O for 5 minutes and leave in PBS for a minimum of 5 minutes.
The tissue is circled with a PAP pen (Sigma Liquid Blocker, Vector ImmEdge Pen or RPI PAP
pen) which creates a hydrophobic barrier to contain the antibodies and blocking solutions. We
use a “Universal Blocking Buffer” (PBS-BB) which contains an excess amount of protein (1%
BSA) and detergent (0.3% Triton X-100) is placed on the slides for 30 minutes. Since most
antibodies are raised in mouse, rabbit and/or goat, this mixture allows multi-labelling.
Prepare a humidity chamber by placing a moistened strip of paper towel in a plastic slide folder
with lid (RPI 247824). The slides will be placed above the paper and filled with Blocking Buffer.
After 30 minutes the BB is tipped off, the excess wicked off as it runs to the bottom of the PAP
circle and filled with primary antibody or no antibody containing PBS-BB. Overnight incubation
of the slides is recommended to occur in the covered humidity chamber in a refrigerator.
Primary antibody is made up at optimal dilution in Blocking Buffer, and a negative control slide
without antibody (and/or normal sera from the host species) is run alongside the test slides.
Negative controls are important in assessing the optimal detection conditions as it will receive
all reagents involved in the detection steps.
In research laboratories overnight incubation of the primary at 4oC is usually used to maximize
the reaction time of the antibody to the antigen. It is possible to substitute higher antibody
concentrations and shorter times if necessary.
DETECTION
The next day the slides are removed from the refrigerator, tipped to remove the antibody and
immediately placed in a container of PBS. Once all the slides have been processed, the PBS is
changed every 5 minutes for 3 times (a rotating table will assist in the complete washing of the
tissue).
Once washed the secondary antibody is added to each slide and allowed to react at room
temperature (RTo) for 1 hour before being tipped off and washed 3 times in PBS (5 minutes
each). Directly conjugated secondary may then be nuclear counterstained with bis benzimide,
and cover slipped.
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Detection using Tyramide Signal Amplification (TSATM) kits
(PerkinElmer Life Sciences)
PROCEDURE:
1. Deparaffinization and rehydration of tissue samples:
a. Soak in Citri-Solv x two chambers (10 minutes and 5 minutes)
b. 5 min in 100% 2-propanol x three chambers
c. 3 min slowly running water
d. 5 min in dH2O
2. Antigen retrieval using 0.01M citrate buffer, pH6.0, 20 min (in a rice steamer, see
procedure) with 30 minutes slow cool down to room temperature.
3. Rinse slides in
a. 3 min slowly running water
b. 5 min of dH2O
c. 5 min of Phosphate Buffered Saline (PBS)
4. Quench endogenous enzyme with 3% (v/v) H2O2 in PBS for 10 min
5. Rinse slides in PBS for 5 min x 3
6. Carefully wipe glass dry around the tissue section with a paper towel, to remove excess
buffer and create a dry area to circle the tissue with a hydrophobic barrier pen (PAP,
ImmEdge or Liquid Barrier)
7. Apply PBS-Blocking Buffer to cover the tissue but not so much it runs off. (about 100200 ul)
8. Tap off blocking solution
9. Immediately apply 70-100 ul primary antibody diluted in PBS-BB to the section and
incubate at 4 C overnight in humid chamber. (To avoid overfilling and subsequent
drying - apply the lesser amount to cover the tissue, wait a minute and add enough to
fill the PAP pen circle.)
10. The next day tap off primary antibody solution and place into PBS.
11. Rinse slides in PBS for 5 min x 3
12. Shake off excess liquid.
13. Apply 100 - 150 ul secondary antibody (DAR-Fitc) diluted 1:200 in PBS-BB to the
section and incubate in humid chamber at RT for 60 min
14. Tap off secondary antibody solution
15. Rinse slides in PBS for 5 min x 3
16. Immerse tissue in Bis benzimide to counterstain the nuclei [1ul (2mg/ml stock) in 10 ml
PBS]
17. Mount in 50%Glycerol in PBS using a 1.5 thick coverslip. Check the staining.
18. To remove coverglass: Place slide in staining jar with PBS.
19. Gently tease off the cover glass.
20. Rinse slides in PBS for 5 min x 3 changes of buffer in staining jars
21. Apply Sheep anti-FITC-HRP 1:200 in PBS-BB for 30 minutes minimum in a humid
chamber at room temperature. (Try 60 minutes.)
22. Wash slides in PBS for 5 min x 3 changes of buffer in staining jars
Note: The following detection systems are very sensitive and require rigourous washing to
control background.
23.Separate slides into 2 detection groups (TSA and TSA-PLUS).
24. For the TSA standard detection group (3 slides = BB, 1:50,000 and 1:100,000 dilution
of Rabbit anti-GFAP). Prepare 735 ul of Amplification Diluent in a 1.5ml microcentrifuge
tube.
25. Immediately before use add 15 ul of Fluorescein Tyramide Reagent and mix well.
(Dilution of 1:50)
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26. Add 125 – 200ul of Fluorescein Tyramide Reagent to each slide. React for 3 minutes.
27. Tip off excess reagent, rinse slide in PBS and then soak in a container of PBS. Repeat
for 5 complete changes of buffer, 5 minutes each.
28. For the TSA-PLUS detection group (3 slides = BB, 1:50,000 and 1:100,000 dilution of
Rabbit anti-GFAP). Prepare 735 ul of 1X Plus Amplification Diluent in a 1.5ml
microcentrifuge tube.
29. Immediately before use add 15 ul of Fluorescein Amplification Reagent and mix well.
(Dilution of 1:50)
30. Add 125 – 200ul of Fluorescein Amplification Reagent to each slide. React for 3
minutes.
31. Tip off excess reagent, rinse slide in PBS and then soak in a container of PBS. Repeat
for 5 complete changes of buffer, 5 minutes each.
32. Immerse tissue in Bis benzimide to counterstain the nuclei [1ul (2mg/ml stock) in 10 ml
PBS]
33. Mount in 50%Glycerol in PBS using a 1.5 thick cover slip. Dot with nail polish if
transporting.
More information and regent recipes and vendors are available at:
http://www.neurosciencecore.uab.edu/coreb_resources.htm . The Molecular Detection Core
can help you obtain detection of antigens of interest to your research, email clatham@uab.edu
or call the core at (205) 996-6556.
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