Cell Signaling Technology (CST) is a different kind of life sciences company-one founded, owned, and run by active research scientists, with the highest standards of product and service quality, technological innovation, and scientific rigor. Founded in 1999 and headquartered in Danvers, Massachusetts, USA, CST employs over 400 people worldwide. We consistently provide fellow scientists around the globe with best-in-class products and services to fuel their quests for discovery. CST is a company of caring people driven by a devotion to facilitating good science-a company committed to doing the right thing for our Customers, our communities, and our planet.
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.
Hi, this varies depending on the cell line. Please refer to information from the cell line supplier on optimal growth conditions for your specific cell line. For mammalian cell lines, 5% is the most common CO2 concentration, but some cell lines may require 7.5% or a different CO2 concentration to properly maintain pH of the media, which is typically buffered with a bicarbonate-based system. By the way, if you are culturing insect cell lines, these will have a lower temperature (again, check with the supplier) and likely do not require maintaining a specific CO2% since the media used for these cell lines isn't buffered with bicarbonate.
Oh sorry about that! Most incubators don't actively adjust humidity levels the way they do for C02; many users place a pan of water in the bottom of the incubator and include a bacterial/fungal inhibitor. This is sufficient to reduce the rate of vapor evaporation from cell culture vessels, as long as they are being consistently maintained and checked.
@@funny11744 A high humidity is recommended (around 95%) to minimize water evaporation from cell culture media. Evaporation can lead to changes in concentration of salts and other components, harming the cells. Opening the incubator door can cause a temporary dip in humidity due to cooler room air entering. This is why it's important to minimize the frequency and duration of door openings.
Thanks for the question! We have used antifungal and antibacterial reagents for our water baths in the past but now opt to clean our water baths weekly, leaving it cleaned and empty on weekends. We also utilize heat conductive metal beads instead of water for our non circulating baths. These will not culture bacteria and fungi like water does and can be easily cleaned with 70% ethanol.
Yes, digital imaging has improved over time, so it is possible to obtain digital images with sensitivity similar to film (sensitivity may vary depending on the imaging system used). Digital imaging is what CST uses currently for visualizing WB results, as it is more eco-friendly and also allows for maintaining electronic records of results.
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.
As mentioned in the video, the optimal sonication settings varies by sonicator, and it can also vary by sample type and fixation method.That's why we don't provide one set of sonicator settings in the video, instead we recommend optimizing. Please refer to Appendix C of the protocol: www.cellsignal.com/learn-and-support/protocols/protocol-simplechip-plus-sonication. Please also feel free to contact our techincal support if you have further questions: www.cellsignal.com/support
The CST Senescence β-Galactosidase Activity Assay Kit (Fluorescence, Plate-Based) #23833 is a quantitative kit, however it is designed for use with cell lines not FFPE tissue. Please contact CST tech support at cellsignal.com/support to inquire if the protocol can be adapted for tissue. They may also be able to recommend other senescence markers that are validated to work in IHC for different sample types.
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?
Hello, what is the purpose of having citrate or EDTA in the buffer? I thought the heating is what is reversing the formaldehyde bond, so it is unclear to be what the role of citrate/EDTA is. Thanks for the video!
Hi, thanks for the question. EDTA and Citrate buffers have different pH (ph 8 for EDTA and pH 6 for Citrate). Some antibody-epitope reactions can tolerate a wider range of pH, but some antibodies perform better with one or the other, which is why we recommend checking the datasheet or online product page for each antibody used. This topic is also mentioned in this video: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-PCayp8kyApU.html, hope that's helpful!
@@cellsignaldotcom Thank you for the answer and link! Recently I tried antigen retrieval with 0.1M citrate at 95C for 30 minutes (increased from 15 minutes), and saw a dramatic increase in non-specific staining and little change in specific staining. Do you have suggestion as to why this treatment might increase background?
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?
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.
Sorry, we don't translate to Arabic, but you could try this browser plugin for Chrome: chrome.google.com/webstore/detail/auto-translate-for-youtub/codommceejgdnbmfednpkhkfnlbepckf
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!
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.
Hi Avana. CST has a summer internship program for undergraduate students and rising high school seniors. Please visit www.cellsignal.com/about-us/social-responsibility/internship-program for information.
There seems to be a time lag from 2016. Nonetheless would like to thank you for putting this up, as an educational video. It's an opportunity to keep up to date. However once uploaded, it was easy to understand, and well explained. It touched upon a number of different aspects relating to the DNA Damage and repair pathway, and the effects of hypoxia. DNA Damage response to drugs, hypoxia on DNA damage. Webinars can provide context. It is always nice to know patient responses since they are at the heart of this, it is about them.
Maybe somebody here can explain why untrained people can substitute continuous moderate workouts with REHIT or SIT workouts while trained endurance athletes do not. The theory is that rapid ATP to ADP creates AMP and AMPK leading to mitochondria biogenesis.
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.
