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IHC Prep & Detect Kit for Rabbit/Mouse Primary Antibody User
Manual
Cat.NO.: PK10019
Description:
The PK10019 kit is designed for developing a complete IHC workflow using primary antibodies raised
in rabbits/mice. The kit contains all the basic reagents, starting from antigen retrieval buffer to
mounting media, that would be required for IHC staining of tissue sections with rabbit/mouse primary
antibodies. By simply optimizing the primary antibody concentration and a few operational
parameters, a complete IHC workflow can be easily established using this kit.
* If you are going to work on mouse samples using a rabbit primary antibody, we recommend you to
use our IHC Prep & Detect Kit for Rabbit Primary Antibody (Cat.NO. PK10017).
What’s included:
Part NO.
Component
Size
HC001
Antigen Retrieval Buffer (50× Tris-EDTA)
100mL
HC002
Washing Buffer (30×)
120mL
HC003
Blocking Buffer
5mL
HC000
Quenching Buffer (RTU)
5mL
HC004
Primary Antibody Diluent (RTU)
100mL
HC005
Polymer HRP-Goat anti-Rabbit/Mouse Secondary Antibody
5mL
HC006
Chromogen Component A
0.2mL
HC007
Chromogen Component B
4mL
HC008
Signal Enhancer
5mL
HC009
Counter Staining Reagent
5mL
HC0010
Mounting Media
5mL
For Research Use Only
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What’s not included:
Rabbit/Mouse Sourced Primary Antibody
Xylene
Ethanol
Pure water (ddH2O)
Coplin Jars or Beakers
Slide Basket
Foil or Material to Cover Beaker
Heating Element
Slide Basket Container
Hydrophobic IHC Pen (Optional)
Thermometer
Wash Bottle
Wet Box
Microcentrifuge Tubes
Pipette and Tips
Cover Slides
Warning:
Use proper protective equipment when working with xylene. Xylene can affect you when inhaled or
upon contact with skin.
• Contact can irritate skin and eyes.
• Inhaling can irritate the nose and throat and cause coughing or wheezing.
• Exposure can cause headache, dizziness, lightheadedness, and passing out. Repeated exposure can
affect concentration, memory, vision, and muscle coordination.
• Use proper personal protective equipment including gloves, protective clothing, eye protection, and
adequate ventilation when handling xylene.
• Dispose off xylene and all kit waste properly.
Tips for using this kit:
To maximize the use of the reagents in this kit, we recommend:
When using the dropper bottles, apply the recommended number of drops onto the slide and use the
tip of the dropper bottle to distribute the liquid evenly across the tissue section. If the tissue is not
completely covered, add 1-2 additional drops of reagent and repeat. If a hydrophobic pen is not being
used, keep slides as flat as possible to prevent the liquid from running off.
For Research Use Only
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Procedure of Building an IHC Workflow
Step 0: Paraffin Removal & Rehydration
1. Prepare slides from tissues sections following routine methods. Label slides with ink that is
insoluble in xylene and ethanol and place slides in basket or coplin jar.
* Use an ink insoluble in xylene and ethanol such as a graphite pencil.
2. Immerse slides in xylene for 20 minutes. Repeat once using a separate container with fresh xylene.
3. Immerse slides in a 100% ethanol tank for 5 minutes. Repeat once in fresh 100% ethanol.
4. Immerse slides sequentially in each of the following ethanol solutions for 5 minutes each:
• 95% ethanol
• 80% ethanol
• 60% ethanol
5. Rinse twice in fresh ddH2O for 1 minute each time.
Step 1: Antigen Retrieval
This kit contains an antigen retrieval buffer based on Tris-EDTA by default, which in our experience is
suitable for over 90% of targets. However, if a negative result is obtained using this buffer, we
recommend trying alternative buffers listed below.
# PR30001 Sodium Citrate Antigen Retrieval Buffer 50x
# PR30014 Protease K Antigen Retrieval Buffer (Heating is not required with this buffer)
1. Use concentrated 50x Antigen Retrieval Buffer (Part NO. HC001) to prepare a 1x solution by adding
the concentrated buffer to ddH2O. For example, to make 500mL of 1x working solution, dilute 10mL
HC001 with 490mL ddH2O.
* 500mL of prepared 1x antigen retrieval buffer in a 1L beaker should be suitable for 1 or 2
baskets. Calculate how much of each buffer will be needed for the experiment to prepare an
appropriate amount of working solution. The working solution has a shorter shelf life than the
concentrate (same hereinafter).
2. Heat 1x buffer solution to 95-98˚C with a heating element.
* Cover the beaker with a lid or foil to avoid vaporizing/vapor inhalation.
3. Place slide basket into heated antigen retrieval buffer. Maintain this temperature while incubating
for 15-20 minutes.
4. Remove the beaker from heat and let it cool to room temperature (takes 35–40 minutes).
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Step 2: Quenching and Blocking
Based on our experience, endogenous peroxidase quenching is not necessary in most cases and can
sometimes even have adverse effects on staining. However, we have included a quenching reagent in this
kit since the quenching step is widely recommended in publications.
1. Use 30x Washing Buffer (Part NO. HC002) to prepare a 1x solution by adding concentrated washing
buffer to ddH2O.
To make X mL of 1x working solution, dilute X/30 mL of HC002 with X-X/30 mL or 29X/30 mL of
ddH2O. For example, to make 600mL of 1x working solution, dilute 20mL HC002 with 580mL ddH2O.
2. Wash slides by rinsing with ddH2O and then briefly immersing in 100 mL of 1x washing buffer.
3. Drain off the liquid on the slides and absorb any residual buffer around the tissue section (make the
slides as dry as possible). Use a hydrophobic IHC pen to draw a circle on the glass slides around tissue
sections (optional).
4. Quenching (Optional): Use the ready-to-use Quenching Buffer (Part NO. HC000) dropper bottle to
add 2-4 drops onto the slide. Leave the slides at room temperature for 10 minutes. Briefly wash the
slide with 1x washing buffer after quenching.
5. Blocking: Use the Blocking Buffer (Part NO. HC003) dropper bottle to add 2- 4 drops directly on
slides, covering the whole tissue and block slides at room temperature for 30 minutes inside a wet box.
* Make sure there are no air bubbles in the blocking buffer on the slide.
* TIP: Use the tip of the dropper bottle to distribute the liquid evenly covering the entire tissue
section.
* Do NOT let the slides dry out.
6. Drain blocking buffer off the slide and absorb any residual buffer around the tissue section after
blocking.
Step 3: Primary Antibody
1. Use the Primary Antibody Diluent (Part NO. HC004) in the kit to dilute your primary antibody (ONLY
primary antibodies raised in rabbits/mice should be used with this kit). It is highly recommended to
test several dilutions of the primary antibody, with a starting concentration of 1~10μg/ml. Apply 80200μL (depending on the size of the tissue) of the diluted primary antibody to the slide, covering the
entire tissue.
* Calculate the volume of the working solution of the primary antibody and use the antibody
diluent to dilute the primary antibody. Preparing too much of the primary antibody working solution is
not recommended until an optimal dilution factor has been obtained. The diluted working solution is
stable for at least 3 months when stored at 4˚C.
2. Incubate at room temperature in a wet box with a lid for 1 hour.
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* Do NOT let the slides dry out.
3. Prepare a 1x washing buffer solution using the 30x Washing Buffer (Part No. HC002) and ddH2O.
Add the prepared buffer to a wash bottle and coplin jar or beaker.
4. Use wash bottle to rinse primary antibody off slides. Then immerse slides in 1x washing buffer for 1
minute. Repeat the rinse and wash steps one more time; drain excess buffer and absorb any residual
buffer around the tissue section.
* If different primary antibodies are being used in the same experiment, wash in separate
containers of washing buffer.
* Use fresh washing buffer for each wash.
Step 4: Secondary Antibody
1. Use the ready-to-use Polymer HRP-Goat anti-Rabbit/Mouse Secondary Antibody (Part NO. HC005)
dropper bottle to add 2-4 drops directly on the slide, covering the entire tissue.
* The secondary antibody provided in this kit will work ONLY when a primary antibody raised in
rabbit/mouse is used in the previous step.
* TIP: Use the tip of the dropper bottle to distribute the liquid evenly covering the entire tissue
section.
2. Incubate at room temperature for 30 minutes inside a wet box.
* Do NOT let the slides dry out.
3. Immerse slides in washing buffer for 1 minute. Repeat two more times using fresh washing buffer
each time.
4. Drain the liquid off the slides and absorb any residual buffer around the tissue section.
Step 5: Signal Development
* Take out hematoxylin and bring to room temperature ahead of the counter staining step.
1. Use pipette to prepare an appropriate volume of Chromogen in a microcentrifuge tube. Ratio of
solutions Chromogen Component A (Part NO. HC006) to Chromogen Component B (Part NO. HC007) is
1:20.
* Depending on the size of the tissue sample, approximately 30-80μl/slide is needed.
2. Use pipette to add enough Chromogen to cover the tissue. Lay slides flat on the benchtop at room
temperature for 5 minutes, or until a brown color develops. If color is visible, stop reaction early by
washing with 1x washing buffer.
3. Use wash bottle to rinse slides with 1x washing buffer. Then immerse slides in ddH2O 3-4 times for
30 seconds each time using fresh ddH2O for each wash. Drain excess buffer off slides and absorb any
residual buffer around the tissue section.
For Research Use Only
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Step 6: Signal Enhancement
1. Use the Signal Enhancer (Part NO. HC008) dropper bottle to add 2-4 drops directly onto the slides,
covering the entire tissue. Incubate for 5 minutes at room temperature.
* TIP: Use the tip of the dropper bottle to distribute the liquid evenly covering the entire tissue
section.
2. Immerse slides briefly in 1x washing buffer to rinse. Drain excess buffer off slides.
Step 7: Counterstain
1. Use the ready-to-use Counter Staining Reagent (Part NO. HC009) dropper bottle to add 2-4 drops
directly on the slide, covering entire tissue. Incubate at room temperature for 2-3 minutes.
* TIP: Use the tip of the dropper bottle to distribute the liquid evenly covering the entire tissue
section.
2. Immerse slides in washing buffer for 1 minute and then immerse in ddH2O for 1 minute. Repeat the
ddH2O wash step two more times using fresh ddH2O for each wash.
Step 8: Mounting
1. Immerse slides sequentially in each of the following ethanol solutions for 5 minutes each:
• 60% ethanol
• 80% ethanol
• 95% ethanol
• 100% ethanol
2. Immerse slides twice in xylene for 5 minute each time using fresh xylene. Drain excess liquid off
slides and place slides in a safely ventilated area (such as a fume hood) to allow the residual liquid to
evaporate.
3. Use the Mounting Media (Part NO. HC0010) dropper bottle to add 1-2 drops directly on the slide, on
top of tissue ensuring the tissue is completely covered by mounting media.
* You can use a clean glass stick to distribute the mounting media evenly before placing the cover
slide.
4. Carefully place cover slides over tissue and mounting media.
* Avoid trapping air bubbles underneath the cover slide.
5. Allow slides to fully dry in a horizontal position for 30-40 minutes.
Step 9: Data analysis and optimization
1. Analyze with a light microscope.
For Research Use Only
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2. If unsatisfied with the results, perform further optimization following the guidelines below:
2.1 If weak or no staining is observed:
• Check whether your primary antibody was raised in rabbit/mouse.
• Check whether the target is expressed in the tissue being analyzed at moderate to high
levels. If that is not the case, refer to publications to include a positive control in your experiment.
• Increase the concentration of primary antibody (up to 100μg/mL).
• Extend antigen retrieval time (up to 60 minutes).
• Try using an alternative antigen retrieval buffer.
If none of the recommended approaches work, it is possible that the primary antibody is not
suitable for IHC.
2.2 If moderate to strong nonspecific signal is observed:
• Check whether the tissue was contaminated with microorganisms during the experiment.
Confirm that little to no blood exists in your tissue sample before slicing.
• Try lowering the concentration of the primary antibody or shortening the primary antibody
incubation time (down to 30 minutes).
• If this kit is applied to mouse tissue samples, background signal might be observed due to
endogenous immunoglobulin being directly detected by the secondary antibody. To overcome
this, try increasing the primary antibody concentration or shortening the secondary antibody
incubation time. It is also highly recommended setting up a negative control without the primary
antibody in this situation. If you are planning to use a rabbit primary antibody for staining mouse
samples, we recommend using our IHC Prep & Detect Kit for Rabbit Primary Antibody (Cat.NO.
PK10017).
• Try shortening the antigen retrieval time (down to 15 minutes).
• Skip the quenching and/or signal enhancement steps.
• Use an alternative antigen retrieval buffer.
If none of the recommended approaches work, it is possible that the primary antibody is not
suitable for IHC.
* Important: DO NOT dilute the secondary antibody using the Primary Antibody Diluent (Part
NO. HC004) provided in this kit.
For Research Use Only
Page 7 of 7
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PRODUCT-SPECIFIC PROTOCOLS
IHC (10883-1-AP)
Sample type
Sample
preparation
Antigen Retrieval
Blocking buffer
Primary
antibody dilution
Incubation time
Signal detection
human cervical cancer
tissue
Paraffin-embedded
Tris-EDTA buffer (pH 9.0)
3% BSA in TBS
1:2000
1.5 h at room temp
Reagents for color
development
PROTOCOL
Deparaffinize and rehydrate
1. Deparaffinize slides in 2 baths of xylene for 20 min each.
6. Rinse slides 3 times with 1X TBST for 1 min each.
2. Rehydrate slides by sequential incubation with 100%, 95%, 80%, and 60%
ethanol, 5 min for each bath.
7. Incubate slides with sufficient peroxidase labeled polymer secondary antibody
for 30 min at room temperature.
3. Immerse slides with distilled water 3 times for 1 min each.
8. Rinse slides 3 times with 1X TBS for 1 min each.
Antigen Retrieval (check table above)
9. Incubate slides with enough DAB for 2-5 min at room temperature till a brown
color develops.
1. Transfer slides to a container and cover with antigen retrieval solution
according to the table above.
10. Rinse slides 3 times with distilled water for 1 min each.
2. Heat slides in a microwave on medium power for 10 min.
11. Incubate slides in a bath of hematoxylin at room temperature to stain the nuclei
for 3 min.
3. Allow slides to cool in the buffer for 35 min.
12. Rinse slides 3 times with 1X TBS for 1 min each.
Incubation with Primary Antibody and Signal Detection
1. Rinse slides 3 times with 1X TBS for 1 min each.
2. Incubate slides with 3% H2O2 solution (diluted in distilled water) for 10
min to quench endogenous peroxidase activity at room temperature.
13. Immerse slides in distilled water for 5 min.
14. Immerse slides sequentially into 60%, 80%, 90%, and 100% ethanol bath for 5
min each.
15. Immerse slides in xylene bath for 5 min, then immerse in another fresh xylene
for 5 min.
3. Rinse slides 3 times with 1XTBS for 1 min each.
4. Block the slides at room temperature for 1 h in Blocking buffer.
16. Mount the slides with a small drop of neutral balsam and add a coverslip. Airdry slides in the hood.
5. Incubate slides with primary antibody (diluted in dilution buffer) for 1.5 h at
room temperature.
17. Examine slides under a microscope.
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