概述

  • 产品名称
    Trypsin Enzymatic Antigen Retrieval Solution
    参阅全部 Antigen retrieval solution 试剂
  • 经测试应用
    适用于: IHC-Pmore details
  • 常规说明
    Protocol Notes: 1) Deparaffinize tissues and hydrate to water. If necessary perform a hydrogen peroxide block, wash in water, and rinse in PBS or TBS. The tissue section is ready for trypsin digestion. 2) Combine 1 part trypsin concentrate with 1 part buffer (working solution stable for 5 working days at 2-8ºC). Incubate section for 5-10 minutes at 37°C or 15 minutes at room temperature. Then, rinse well in PBS or TBS buffer and proceed with immunostaining procedure. This product utilises a color-coded pH indicator (rose) for easy identification. The end-user can visually inspect the solution and see that the mixture or buffer is at the proper pH. If the mixed solution is purple, the pH is too high for optimum digestion. If the buffer or trypsin solution turns orange or yellow, the pH is too low for optimum digestion.


    Trypsin is a commonly used digestive enzyme. In formalin-fixed, paraffin-embedded tissues, certain antibody protocols required enzyme pretreatment for proper immunohistochemical staining.

性能

应用

Our Abpromise guarantee covers the use of ab970 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
IHC-P Use at an assay dependent dilution. Ratio for 25ml concentrated trypsin and 25 ml buffer is 1:1 - 1:3.

文献

This product has been referenced in:
  • Choi MC  et al. Alleviation of Murine Osteoarthritis by Cartilage-Specific Deletion of I?B?. Arthritis Rheumatol 70:1440-1449 (2018). Read more (PubMed: 29604191) »
  • Hong HN  et al. Cancer-associated fibroblasts promote gastric tumorigenesis through EphA2 activation in a ligand-independent manner. J Cancer Res Clin Oncol 144:1649-1663 (2018). Read more (PubMed: 29948146) »
See all 8 Publications for this product

客户评价及客户问答

1-10 of 12 Abreviews or Q&A

works well

Excellent Excellent 5/5 (Ease of Use)
Abreviews
The trypsin digestion worked well for the PRG2 (ab14462) staining.
The enzyme working solution is easy to create. The corresponding buffer is included in the kit.
For the digestion it takes a maximum of 15 minutes.

Abcam user community

Verified customer

提交于 Feb 08 2018

Answer


There are 2X 25 ml vials in the kit

1 vial is 25 ml Trysin (concentrated liquid)

2nd vial 25 ml Trypsin buffer

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Answer

Thank you for contacting us.

For a standard IHC-P protocol I usually recommend a HIER method (Heat Induced Epitope Retrieval) for the antigen retrieval step using a Tris/EDTA pH 9.0 buffer as described in section "D" of the following protocol:https://www.abcam.com/index.html?pageconfig=resource&rid=11384.

However you could also use an enzyme mediated antigen retrieval step and using ab970 for this should work fine. Please let us know if you have problems as these products are covered by our abpromise guarantee.

At Abcam, providing quality service is just as important as providing quality products. Our Abpromise® ensures that you can trust our products to perform as stated on the datasheets, or we will offer a replacement, credit, or refund.

The Abcam Abpromise® guarantee:
- 100% Scientific and Customer Support for any product you buy from Abcam or one of our authorized distributors.
- We guarantee our products work in the tested species and applications stated on the datasheet.
- We will replace or refund products not performing as stated on the datasheet if reported within 6 months of purchase.
- We investigate any quality concerns raised by customers to ensure our catalog contains products that perform to the highest standards.

Please note these conditions to our Abpromise®:
- Protocol information must be provided in order for the claim to be validated.
- Antibodies tested in recombinant samples only are not guaranteed for use on endogenous samples.
- “Predicted to react” information on the datasheets is provided for reference only and these species are not guaranteed.
- Fast Track antibodies are covered for ELISA against the immunizing peptide only.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Answer

Thank you for contacting us. Yes, ab970 comes with 25 mL of concentrated Trpsin and 25 mL of Trypsin buffer.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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https://www.abcam.com/abreviews

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Answer

Thank you for your email.

To answer your questions and make some comments/suggestions that hopefully will help you further:

1) The antibodies (shipped as is) should be stored aliquoted and stored at -20 or -80 degree. This would also apply to the 1/10 stock solution as well. Please make sure to avoid freeze-thaw cycles. For ab6994, a brief period of storage at +4 degree for 1-2 weeks would be fine, but for long term storage -20 or -80 degree is recommended.

2) For ab6994, you should have received 100 ul, and for ab104225 you should have received 50 ul. You can spin the vials briefly before opening and the liquid should be at the bottom of the tube and it would be visible. In thecase of these 2 antibodies, diluting them to 1/10it might be OK as they are either whole antiserum (contain other proteins) or contain lots of BSA as stabilizer.
In general, it is recommended to dilute the antibody to the working solution just before use.

A 1/100dilution would be an increase in antibody concentration compared to 1/500 (which is more dilute), as 1/100 contains more antibody than 1/500.

3) Yes, the higher temperaturewill help with the antigen retrieval.

4) Yes, this is probably fine. But in case of no signal, you might consider reducing the time to e.g. 2x 30 sec.

I'm glad to hear you are getting now signal with the Fox 3 antibody. As for the VWF antibody, would also use more primary and secondary antibody, increase the retrieval temp to 37 degrees and reduce the wash time. Yes, checking the reference could provide additional helpful information.

I wish you good luck and look forward to hear back.

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Question

The trypsin antigen retrieval did not work. I'd like to review what I did to see if there are any large errors.

Rats wre perfused in situ and brains were preserved in 4% paraformaldehyde solution overnight. We purchased the paraformaldehyde commercially pre-prepared. We then sucrose protected the brains (after speaking with an abcam rep) in a sucrose series to freeze protect them. We then froze the brains for a period of about a month.

I recieved the Fox-3 antibody from abcam as a 50ul concentrate. In reading the antibody reviews online I saw that our target antibody concentration would be between 1:500-1:2000. So I initially added 500ul to the 50 ul concentrate to have an initial 1:10 dilution (roughly). Then I took 1 ul of this working solution and diluted it with 200 ul PBS to get my 1:2000 dilution. I followed this with 1ul working solution in 100 ul PBS to get my 1:1000 dilution. Then, 2:100ul PBS to get my 1:500 dilution. I did this because an abcam representative said I should shoot for 100ul per slice.

I took my test slices, washed them in PBS for 5 minutes, then outlined them in a histology pen, and applied the primary. I spoke with an abcam rep last week who advised us not to block unless we had too much non-specific staining. I used the trypsin enzymatic retrieval kit you recommended which entained mixing the trypsin concentrate with a diluent included in the kit at a ratio 1:1. I applied 100ul of this per slice for 15 minutes at room temperature under a fume hood (not in the dark--this was not required in the literature). I then washed twice with PBS, and I applied the primary on a counter top at roomtemperatureand then stored in at 4 degrees c overnight in a dark container. After this, I rinsed the slices in PBS, and thenbathed the slices twice in PBS to get the primary off, and then applied the Dylight secondary (dilution info below) for one hour at room temp in a dark environment. I then washed once with PBS and applied the DAPI mounting medium we purchased from abcam. I coverslipped the slides and left them for about an hour at 4 degrees c in a dark slide holder. I then attempted microscopy.

Secondary info: (Donkey anti rabit Dylight 488) from its stock solution and read online that a 1:50-1:200 dilution would be optimal so I tried 1:100 which should have produced at least some fluorescence. I took the equivalent of 1ul Dylight to 99ul PBS done in the dark..

The VWF antibody was prepared the exact same way except the suggested concentrations were slightly different.
I took the 100ul concentrated antibody that abcam shipped and immediately added 900ul for a 1:10 working solution dilution. Then I did an experiment with 1:200 (5ul working solution in 95ul pbs); 1:500 (2ul working in 98 ul pbs); and 1:800 (2 ul working in 198 pbs). Secondary was applied at same conc.

Where should we go from here?

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Answer

Thank you for your email.
I am sorry to hear the antigen retrieval step did not improve your results.

I have looked throught your protocol you sent me and have the following suggestions, comments and questions:

1) You are making a 1/10 stock solution for each antibody. How do you store the antibody afterwards?

2) Also, in my understanding, diluting the entire vial of antibody - although it makes it easier for pipetting - can lead to a loss of antibody to the tube wall due to absorption andan increase in volume. Thus, increasing the primary antibody concentration might be necessary(e.g. 1/100).

3) For the enzymatic antigen retrieval, a temperatue of 37 degree would be recommendedas this is the temparature where the enzyme is most active.

Here is the link to our antigen retrieval protocol page: https://www.abcam.com/index.html?pageconfig=resource&rid=11488. In section 3, you can find information about the enzymatic retrieval.

4) In your protocol description, you mentioned: "I rinsed the slices in PBS, and then bathed the slices twice in PBS to get the primary off". How long did you bathe the slides in PBS each time? If this is done for too long, you might potentially also lose primary antibody.

5) As for the secondary antibody, it will only bind specifically if the primary antibody is present. Thus, doing the antigen retrieval at a higher temperature and using potentially more primary antibody should help with that. In addition, you might also want to try a lower dilution and longer incubation time with the seconadry antibody (e.g. 1/50 for 2 h).

6) You are using perfused frozen sections which we abbreviate IHC-FoFr.
For the VWF antibody (ab6994) this application has been tested, and it was added based on the following publication:
Fujimoto KL et al. Naive rat amnion-derived cell transplantation improved left ventricular function and reduced myocardial scar of postinfarcted heart. Cell Transplant 18:477-86 (2009). IHC-FoFr; Rat. PubMed: 19622235
It might be worthwhile checking this publication for additional protocol details or contacting the authors for the details of their technique.
In addition, a customer submittedthe following Abreview for IHC-FoFr:
https://www.abcam.com/index.html?datasheet=6994&tab=abreviews&intabreviewid=11467

As for the FOX3 antibody (ab104225) this application has not been tested yet, and we do not have information if and how well IHC-FoFr would work with this antibody. Thus, we also do not havea specific protocolestablished for this antibody. Since IHC-P was tested with ab104225, IHC-FoFr might work as well. Here is the link to the Abreview a customer submitted for IHC-P:
https://www.abcam.com/index.html?datasheet=104225&tab=abreviews&intabreviewid=25847

I have also attached a paper describing how heat-inducedantigen retrieval can be used on frozen sections.

I hope this information will be of help to you. Please let me knowif these suggestions are improving your results or not.

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Answer

Thank you for your reply.
I'm glad to hear that the secondary antibody shows fluorescence.

As for selecting another secondary antibody, could you please let me know which fluorophore or wavelength range is covered by your miscroscope? Based on that informationI can then select a different antibody for you.

Please let me know. Thank you!

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Answer

Thank you for your email.
I would recommed one of the following trypsin products:
ab970: Trypsin Enzymatic Antigen Retrieval Solution
https://www.abcam.com/trypsin-enzymatic-antigen-retrieval-solution-ab970.html
ab64205: Trypsin Enzymatic Pretreatment Kit
https://www.abcam.com/trypsin-enzymatic-pretreatment-kit-ab64205.html
ab128214: Trypsin (Enzymatic Pretreatment)
https://www.abcam.com/trypsin-enzymatic-pretreatment-ab128214.html
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for contacting us.
I am sorry to hear that the antibodie might not be working as expected.

As we discussed over the phone the following troubleshooting tips might help in finding the cause of the problem:

1) Try antigen retrieval (enzymatic) using one of the products we discussed (ab64201, ab64220, ab64205 or ab970).

2) Check if secondary antibody works (e.g. with fluorescent plate reader or fluorescence micrscope, or with another primary Ab)

Please let me know if and how your results improving.
I wish you good luck and look forward to hear back from you.

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Answer

Thank you for contacting Abcam. For ab6328, I would recommend using ab970, the Trypsin Enzymatic Antigen Retrieval Solution that you mentioned. I believe that this will give you very good results in your IHC staining. For ab1416, from what I have read, it look as though that heat mediated antigen retrieval is recommended. You could use ab972, the Heat Mediated High pH Antigen Retrieving Solution, the protocol for this product is available on the website. If there is anything else I can help you with, please let me know.

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