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hello everyone my name is Josh Nick Bell and I would give you today an introduction to some optimization steps and guidelines you need to put in place your multiplexing as a using the Opel technology as as you may know already opal is a way to look at several biomolecules today up to eight biomarkers on a single tissue section while retaining morphological context so it's a way to look at for example the tumor microenvironment and the immune systems the image is there around the tumor and quantify signal in case you want to score different checkpoint or biomarkers we have now a lot of recommendations and guidelines and tips to multiplex in the best and easiest way as possible and as you know you don't have to worry with opal about the species of your primary because you can work with different primary antibodies made in the same species so two is the process of developing your seven and nine core oh I see we provide opal detection kids and those kids contains everything you need to perform your essays so we have chosen the best combination of dyes and also solution such as the secondary antibody which is a mixture of anti mouse and anti rabbit IgG except for the new mouse kids where it's just anti rabbit antibody and also all the solutions in terms of blocking solution antibody different amplification solution and so on so you don't have to worry about making your one buffer so opal is a rebel addition of sequential staining cycles and every every cycle include a blocking step a primary antibody incubation which is usually just 30 minutes secondary antibody introduced in introduction with conjugation 2 h RP h RP will make the deposition of opal in just 10 minutes and then we strip the antibodies and we start a new staining cycle so the staining cycles are every time quite short which enables you to do several staining cycles in one day but as it could be a long process manually we now have also a way to fully automate opal using the like a bond and using the wash Discovery Utah and now for example is the like a bond you can do your 7 color as they in 12 hours so you can do it overnight and also with better reproducibility because we have looked internally and I mean doing some reproducibility and validation tests and we usually have a CV below 15% between 1 and between works so today I will cover in I hope a quite short video the mono plaques optimizations before moving on to your multiplexing and looking at balancing your signals and so on to have to have a very reliable multiplex assay so the first thing which is quite important is to prepare your slides carefully so we recommend to bake slides in the oven at 55 or 60 degrees overnight before performing sex I lean and a sternal gradient bath two completely deprived in eyes your tissue sections for those working manually and using the microwave and having dedicate tissue section you could use ten percent not hold buffered formalin for ten minutes to fix the tissue section onto the glass lights that would really help to maintain the tissue section onto the slide over the several stealing stealing cycles so the first step when you start reason with a new panel and with new antibodies is first to validate each antibody and we like to do a short iteration so we if you have any dated your antibody already using da be so standard RC we highly encourage you to use the same DB concentration I mean the same primary antibody concentration as you use for da B but in case you're going to dilute your antibody a bit further you can do a two time or full time dilution you can also try pH six versus pH nine although most antibodies work really well users usually with pH six or after several insertion with reverse steps really good thing using inform and looking at your opal stinging is you can now reproduce you can mimic a DB staining from your fluorescent tissue section from your fluorescent image so as you can see here if I look for example at opal 520 you know fluorescence I can reproduce a DB staining where the Dappy would be like hematoxylin and the opal 520 is represented as DB staining we call it a pathology view and this way you can really compare what you have using open with what you get what you used to have using DB so first what I look is I look at the staining pattern I want to make sure I get a very nice staining pattern so for example here with cd8 we know it's a membrane staining and I want to make sure I have a nice wing and that all the antigens are well saturated if it becomes putty or patchy I know I went too far into the direction and I need to concentrate a bit more and vice versa if I see it's really chunky or if I have really high signal I know have some room for dilution with inform and multispectral imaging you can now really quantify your signal and and subtract autofluorescence so now you can have a really good idea of your signal to background and we recommend to have a signal to background of at least 10 so you can look by going over by hovering onto the image you can you can look at signal and you can just look at signal specific signal versus background and make sure you get the best signal to back on as possible so if you're still unsatisfied after this titration with the staining pattern maybe you need to try different antigen retrieval method or maybe try longer incubation time of your primary so I recommend to start with 30 minutes but if you need to go up to an hour even maybe overnight feel free to do so although I believe overnight incubation just brings both background and it's often not necessary in terms of blocking solution as I say we provide we provide blocking solution in our kit but feel free to use also blocking solutions if you have to reduce the background or if you feel you have nonspecific staining and of course if you're still not happy feel free to try another primary antibody today we have many providers and clones available so you can you're flexible in that way ok do your controls I always repeat that but it's quite crucial when you start with new antibodies and with the new stripping and with a new stripping metal method to make sure that your stripping is complete and we have two approaches for that so the first one is to do a full staining cycle using for example open five twenty then you strip your antibody using your preferred stripping method and you come again with just a secondary and another type so let's say Oh pal six twenty and if the stripping isn't complete you would see some leftover of signal like who you can see on the image on the right you see some of the other guy co-localize with the first woman that means the stripping was not complete and the secondary is buying - some leftover of the primary another way to do is if for example you're looking like here at cd8 membrane marker then you can perform the seven second staining cycle after stripping using a nucleon marker if the stripping is complete you shouldn't see any colocalization between the membrane marker and the nuclear marker I know that you know all application guides you we highly recommend microwave but feel free to use any stripping methods you have in your lab such as water baths so for example for water baths you can use 95 degrees for 20 minutes or 98 degrees if the stripping is nice not enough you can use different kind of microwaves pressure cooker and also we have some news using that Kapiti link in order to perform heat treatment for four on the slides for the stripping and of course we also use the lake a bond and Ventana Discovery Utah so again doing your control is crucial especially if you want to publish and so it's quite important to do a no primary antibody control and also a nicer type control so once you have when once you're satisfied with what you get in terms of staining pattern and signal intensity it's quite important to define the order of addition and the best method I found to look at how your antigen is going to react to several heating steps is using Swissair section one section that I would stand after one staining one heating heating treatment one cellular section that I would stain after three heating steps and one that I would stain after six heat treatments every time between every heat treatment I let it cool down I wash I use a fresh antigen with Revolt solution again before repeating the heat treatment and this way you can really see how your how your your epitope is going to react through several with levels we have found that about seventy even more 80 percent of antibodies are actually giving a better staining pattern and signal after several heating steps so we believe hitting those sequential heating steps are not affecting the tissue but it's it's it's true that for a few maybe 10% epitopes we could see a decrease in signal like if the epitope was degraded so in that case you know that this antibody has to be placed first in the sequence and if you have an increase in staining pattern or an increase in signal you know that this one could be placed at the end so I really like during this experiment it takes one day but it really really gives you a lot of good information about where to place your primary antibody in the sequence the good thing with opal is that you don't have to worry about the opal dye stability we have found that the whatever the dyes they are quite stable throughout the whole the whole staining push it procedure so even if you do one three or six stripping steps the signal will stay stable the cause was six stealing cycles so you don't have to worry about which one to put first the only thing that you can pay attention to is how you would pair your opal dyes to your to your markers so we have assessed on the Vectra Polaris but also on the vector three the difference in intensity of our different dyes so personally true of the dye and also the spectral response of the camera we have found that opal 690 and opal 780 always the lowest the dimmer signal so I would encourage you use this opal 690 and o opal six 780 with the most abundant marker you have so for example say to carotene or c d3 is something where you have usually a strong strong signal also if you're looking at co-localize markers we recommend to pick dyes that are not spectrally close so for example if you look at CD three and cd8 and pd1 don't use five twenty five forty and five seventy you can use five twenty six twenty and six ninety we have enough room so that you can separate your diet for your co-localize markers and once you have done just a few optimization steps you're not you're now ready to do your multiplexing and and with multispectral imaging we like to balance our signals so when you look at info and idea of multiplexing experiment multiplexed images you can you can look at signal expression and balance the signal intensity by just adjusting the opal dye dilution so it's very easy you just have to dilute a bit more if the signal is very strong or you have to increase the concentration a bit if you want to increase the signal but you don't have to play on the antibody concentration and so on again and that's it that's how you can have this beautiful multiplexed image and using inform you can also look at every component and check again that you don't have any on specific collectivization and that your unmixing is perfect so the take-home message is really keep it as simple as possible and improves your staining step by step and if you follow those steps you will have a multiplex taxi in a very fast fast way thank you for your attention and talk to you soon