Immunohistochemistry Protocol for Paraffin embedded Tissue Sections

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8 жыл бұрын

IHC Protocol Video for Paraffin-embedded Tissue Sections from Cell Signaling Technology (CST)
👉 CST Protocols: cellsignal.com/protocols
👉 Download the Guide to Successful IHC: learn.cellsignal.com/cst-appl...
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👉 Technical support: cellsignal.com/support
Immunohistochemistry (IHC) is a powerful microscope-based technique that uses an antibody to view a specific protein in biological tissue. It is often used to diagnose abnormal cells in solid tumors, visualize molecular markers for cellular events like apoptosis, and monitor the localizations and expression of biomarkers. However, individuals new to this method can struggle due to the many steps in the procedure that can each add variations that will impact staining.
Cell Signaling Technology (CST) scientists have determined optimal conditions for CST antibodies developed and validated in-house for IHC. In this video, we demonstrate the IHC protocol we have optimized for formalin-fixed paraffin embedded (FFPE) tissue sections so you can replicate results in your laboratory and obtain consistent results.
We will first describe how to prepare your sample and deparaffinization/rehydrate the FFPE samples followed by the antigen unmasking step, the most difficult step in an IHC protocol. Next, we will review chromogenic staining with the SignalStain DAB Substrate Kit followed by how to mount sections to a coverslip before viewing on a microscope. Finally, we describe how using different conditions than those recommended each antibody Product Data Sheet, like changing the recommended antibody diluent, can affect your final results.
If you’re interested alternate protocols including performing IHC on frozen tissue samples visit the Protocol at the Cell Signaling Technology website (cellsignal.com/protocols).

Пікірлер: 62

@user-dy3rx7vq4z Жыл бұрын

A laboratory technician from Iraq, and I work on these analyses. Thank you

@manuelpv7894 Жыл бұрын

This video is gold. Thank you.

@xchg_107 4 жыл бұрын

Thank you So much for this helpful video !

@anjaliaggarwal3052 2 жыл бұрын

Very precisely conveyed, thankyou

@MinaAzer 11 ай бұрын

Nice ! Thanks for this amazing video.

@dhanamjai4747 3 жыл бұрын

Thank you, very helpful.

@tijesupemibabatope9058 Жыл бұрын

Hello, i would like to know the difference between peroxidase and protein block I am a little confused

@mrshikari2 3 жыл бұрын

Thank you, this is very helpful :)

@farzanehtaherian3783 7 ай бұрын

Excellent, please explain how can use PR-ER and her-2 tumor marker?

@sadiasarwar8627 5 жыл бұрын

very helpful. Thanks

@bouhnikdjalil7094 5 жыл бұрын

Thank you so much

@sigfridshayo4715 5 жыл бұрын

very useful indeed

@akanjiomotosho5186 6 жыл бұрын

Educative video. Thanks for sharing.

@VibrantVitaHealth 6 жыл бұрын

Thanks

@user-mh9lz8et2p 7 ай бұрын

Well explained

@d7132 Жыл бұрын

Hi, thank you for this beautiful demonstration for IHC. I have a question concerning dehydration and mounting process, proir to xylene should I let the slides to dry from alcohol? or immediately I take them to xylene while they wet?

@cellsignaldotcom Жыл бұрын

Hi, sorry for the delayed reply. Generally, it's advisable to avoid over-drying the slides. You can remove excess drops of xylene by tilting the slide(s) as shown at 2:13 and 2:22 in the video before transferring to EtOH.

@amittirpude 6 жыл бұрын

thanks !!!

@goharrehman7710 3 жыл бұрын

I have to do and learn this please

@saramomo1890 3 жыл бұрын

Thanks for this video can i ask about the pencil uesed for detrimin the borders of section what's the name of it?

@cellsignaldotcom 3 жыл бұрын

Hi Sara, if you are referring to the step at 4:22 in the video, that is a hydrophobic pen. CST does not sell these but if you search for "hydrophobic pen IHC" you should be able to find information.

@goharrehman7710 5 жыл бұрын

Please we need to write it and think what is done

@biomixchannel1235 Жыл бұрын

Good information

@IScreenshotNFTs 3 жыл бұрын

If you immerse in xylene for dehydrating step before mounting, wouldn't the oxidized chromogenic substrates be removed??

@cellsignaldotcom 3 жыл бұрын

Thanks for the question, Josh. The dehydration step prior to mounting doesn't remove deposited chromogens, since these are insoluble to xylene, an organic solvent. Also, as noted in the protocol, the pre-mounting dehydration steps are only 10 seconds each, much quicker than the 5 minute xylene washes used for deparaffinization earlier in the protocol.

@parksibel4620 4 жыл бұрын

Hello, thank you so much for yhe video, Can you please tell me whtas the purpose from boiling in a microwave

@aakashnhoor185 4 жыл бұрын

One of the limitations of IHC is epitope masking (protein cross linking due to formaldehyde fixation) this is overcome by heating

@cellsignaldotcom 4 жыл бұрын

Aakash is correct - an epitope retrieval step is required to make the epitopes accessible to antibodies. We cover this step in more depth in this video: kzfaq.info/get/bejne/sNKepJCH2cDLaas.html

@harithsaad4342 Жыл бұрын

I'm just wondering if DAP brown stain can be removed after counter stain? I feel like when I add DAP the tissue goes brown but after counter stain with hematoxylin the become totally blue ... also I'm new to IHC so just wondering if DAP stain can be easily be gone!

@cellsignaldotcom Жыл бұрын

DAB is oxidized by HRP, and becomes deposited in the tissue, once deposited it is generally not removable. Please visit the links in the description for more IHC resources, or if you have other technical support questions.

@christinaortiz4196 3 жыл бұрын

When you wash the slides in the same buffer multiple times, is it necessary to change the buffer in the box or to have a whole other box filled with the same buffer and ready ?

@cellsignaldotcom 3 жыл бұрын

Hi Christina, I just checked with scientists on our IHC team and they said either method should work. The point of emphasis is to ensure the slides don't dry out. So if you decide to re-use the same container for buffer exchange, just have the buffer prepared in a bottle and ready to go, and you'll be able to exchange quickly.

@christinaortiz4196 3 жыл бұрын

@@cellsignaldotcom Thank you so much for the quick response!

@dichen9626 5 жыл бұрын

A small suggeustion: I think the video quality should be improved. The video resolution is only 360P. Anyway, it is quite great video. Thanks.

@cellsignaldotcom 5 жыл бұрын

Thanks for the suggestion - our newer videos are being produced at higher resolution :)

@PharmaTales 2 жыл бұрын

Can you tell me the protocol followed before tissue embedding in paraffin wax??

@cellsignaldotcom 2 жыл бұрын

Sorry for the delayed reply. This can vary depending on the sample type. We don't have a protocol for tissue fixation and embedding on our website, but if you are using a CST antibody for IHC, you can contact a scientist using the form at cellsignal.com/support. If you include what tissue types you will be using, they may have some information.

@fatimaalsaray9684 4 жыл бұрын

Please can you tell me why we have to use positive charge slides please I have exam in it🥰

@cellsignaldotcom 4 жыл бұрын

The charged surface helps tissue stick to the slide. This may be accomplished by treating the glass surface with poly-lysine and rinsing prior to mounting the tissue sections.

@fafy4_4_22 4 жыл бұрын

Why you use xylol in sectioning what it is benefit ?! Please answer me 💜

@cellsignaldotcom 4 жыл бұрын

As noted in the video, xylene is used to remove the embedded paraffin from the tissue section after it is mounted on the slide. Ethanol washes complete the deparaffinization and remove xylene, and subsequent wash steps in water re-hydrate the section. Deparaffinization and rehydration are required to allow antibodies and counterstains to penetrate the tissue. You can view the full IHC/Paraffin protocol at cellsignal.com/protocols

@fafy4_4_22 4 жыл бұрын

Cell Signaling Technology, Inc. Ok , thank u so much ✨🎀

@dragoniteelcartero5917 4 ай бұрын

Hello, I keep getting non-specific membrane stain in normal breast ducts in HER2 immunohistochemestry, any suggestied modifications?

@cellsignaldotcom 4 ай бұрын

Hi, if you haven't already, please contact technical support (cellsignal.com/support) to get in touch with an IHC specialist who can advise about the specifics of your protocol and tissue.

@noormohammed5126 Жыл бұрын

Hi how I can made serial dilution 10/ 20/50/100 for primary antibody with great thanks

@cellsignaldotcom Жыл бұрын

The CST protocol for IHC does not include serial dilution of primary antibodies. Please refer to the product datasheet for recommended antibody dilutions and diluent buffers for CST antibodies. Are you asking about something else?

@laurenbrown5035 Жыл бұрын

How do you maintain sub-boiling temperature in the microwave?

@cellsignaldotcom Жыл бұрын

Hi Lauren, this depends on the model and power of your lab microwave. You can set up a mock experiment without tissue samples in order to optimize the settings for your microwave. First, find the power and time needed to achieve a boil. Then, perform trials of varying microwave power, and observe the buffer as it is being microwaved. You want to see a low boil with some bubbling, but not boiling over. You can confirm by measuring the temperature, ideally it should be about 95°-98°C. If you have further questions, please get in touch with Tech Support at the link in the description.

@mr.rampachauri9350 2 жыл бұрын

What is the simplify mean of endogenous peroxidase activity

@cellsignaldotcom 2 жыл бұрын

This is referring to expression of peroxidase enzyme(s) by the tissue. Different tissues may have higher or lower endogenous peroxidase. Because IHC chromogenic detection uses (exogenous) horseradish peroxidase (HRP) activity, the endogenous peroxidases have to be blocked to reduce non-specific deposition of chromogen. This is the purpose of the hydrogen peroxide step.

@mr.rampachauri9350 2 жыл бұрын

@@cellsignaldotcom Thanks a lot i got it

@DoctorJPTV 8 ай бұрын

Our tissue sample kept slipping off the glass slide. What should we do to prevent it from happening?

@cellsignaldotcom 8 ай бұрын

If you haven't already, try using charged slides. Some labs prepare these by treating glass surface with polylysine, or you can buy already charged slides from several suppliers. If you have further questions please contact one of our IHC scientists via cellsignal.com/support.

@goharrehman7710 5 жыл бұрын

Hi mam What is charged slide How would charged these slides because they in solater

@cellsignaldotcom 5 жыл бұрын

"Charged slides" refers to glass slides treated with a cationic polymer such as poly-lysine (PLL) or poly-d-lysine (PDL). You can either purchase pre-coated slides, or treat plain glass slides with a PLL solution.

@dichen9626 5 жыл бұрын

@@cellsignaldotcom Thanks for your answer. I have the same question when I watch the video. Now I got it.

@dichen9626 5 жыл бұрын

@@cellsignaldotcom I have got another question here. Is it a must to use charged slides?

@cellsignaldotcom 5 жыл бұрын

@@dichen9626 Thanks for the question, we recommend using slides with charged surfaces because it will help the tissue to adhere and prevent loss of sample during incubations and wash steps.

@goharrehman7710 4 жыл бұрын

still not clear

@cellsignaldotcom 4 жыл бұрын

Hi Gohar, if you have a question about using a CST antibody in your IHC experiment, please visit the link below to get in touch with one of our scientists. www.cellsignal.com/contents/resources-technical-support/immunohistochemistry-technical-support/resources-tech-ihc