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Пікірлер
@lotharmayring6063 2 күн бұрын
tissu can be fixed before frozen or within the freezing process appling crygel with glutaraldeyd
@damienharkin 19 сағат бұрын
Yes, tissue can be fixed prior to frozen sectioning but since fixed tissue retains more water, you will likely need to increase your sectioning temperature to reduce hardness.
@lotharmayring6063 12 сағат бұрын
@@damienharkin increasing the temperature is no problem. But you can form the crosslinking with ex. glutaraldeyd while forming the stabilising gel-red while freezing the probe, there is also a concentration effect on the aldeyd while freezing. But remember with fixation you allways introduce artefacts
@mikephelan5940 6 күн бұрын
Excellent video
@damienharkin 5 күн бұрын
Thanks Mike! Am glad that people are still finding this video useful.
@JULISSALENKAMENDOZAAQUINO 16 күн бұрын
podria utilizar solo hidroxido se sodio ? es necesario que use amoniaco tambien ????
@JULISSALENKAMENDOZAAQUINO 16 күн бұрын
hay manera de sustituir el permanganato como agente oxidante ?
@farzanazargar264 Ай бұрын
Superb... Kindly make a video on congo red Staining
@AdifahAmani Ай бұрын
When i am doing H and E staining, most of sections are washed off during the process.... How to troubleshoot this?
@damienharkin Ай бұрын
It sounds like you are not baking your slides in an oven after cutting sections. You need to place slides in an oven to bake for 30-45 minutes prior to de-waxing and staining.
@NHamidi Ай бұрын
Thank you for making this very detailed video full of trouble shooting points
@damienharkin Ай бұрын
Am glad that you found it useful.
@NHamidi Ай бұрын
The job adverts ask for cutting 30-40 blocks per hour , they need to watch this video 😂
@damienharkin Ай бұрын
That sounds like a lot, but many blocks only require a few sections to be cut.
@georgechandler4664 Ай бұрын
Wow! Beautifully done.
@damienharkin Ай бұрын
Thank you very much!
@T.J.1033 2 ай бұрын
How is the frozen section held across the blade? like in paraffin sectioning you have the wax covering the entire specimen, giving it a rectangle shape, since no such embedding is done in cryotomy, do you just place the whole thing in the holder?
@damienharkin 2 ай бұрын
The section is held together owing to the frozen water present within the tissue. The ice is the equivalent of paraffin used in regular microtomy.
@NaimaMohamed-h2y 2 ай бұрын
This is the best Kohler illumination video ever!! so Helpful Thank you Damien
@damienharkin 2 ай бұрын
Glad it was helpful!
@williamjiang9584 2 ай бұрын
The instructor shows some good techniques here. With case of failure, you can learn the way of improvement. The equipment is new, also different from other labs. Nice demo.
@PR-nf5ot 2 ай бұрын
Hi. Thanks for this video. I am working with spheroids from cell culture . What is your suggestion for thickness? Thanks
@damienharkin 2 ай бұрын
Spheroids can be very tricky, especially when small. My suggestion would be to suspend multiple spheroids in agar following fixation and before processing into OCT. You will also need to be careful when sectioning but embedding multiple spheroids per block should ensure that you achieve cross sections through the middle.
@damienharkin 2 ай бұрын
5-8 microns would be a good starting point for thickness.
@MahmoudadamMahmoudadam 3 ай бұрын
طريقة الكومنت كيف اقدر اعملو وعلي ااااي اساس اعمل كومنت
@chrissygarvey2829 4 ай бұрын
how much time for the 1.5% tartrazine? it wasn't specified!
@damienharkin 4 ай бұрын
1 to 2 minutes should be fine.
@dr.anjaliyadav6896 4 ай бұрын
Can we follow this photoshop editing, what if, journal reject this editing???
@damienharkin 4 ай бұрын
This minor level of editing is acceptable if control and test images are all treated the same way without bias.
@shirishabachigaari8177 4 ай бұрын
Can you do a vedio on alcian blue pas stain please
@damienharkin 4 ай бұрын
This has been covered in my videos on staining for mucosubstances.
@damienharkin 4 ай бұрын
See this video. Mucosubstances - Part 2 kzbin.info/www/bejne/ZpnNYWl3n85gpNU
@milogolding1499 5 ай бұрын
Professor Harkin, in the experiment, did you use pure hematoxylin or a dilution of it? If it was a dilution, what was the dilution ratio that you used? Thank you for your wonderful video!
@damienharkin 4 ай бұрын
We use our Mayer’s Hx undiluted for around 10-15 seconds following application of PAS stain, then “blue” with dilute ammonia.
@hebaallahahussein2280 5 ай бұрын
i am using one from abcam, can i use it on OCT sections?
@damienharkin 5 ай бұрын
Yes, I’ve performed Van Gieson stain on frozen sections.
@sana._.3171 6 ай бұрын
Thanks for this video, Sir I want the interpretation for my lab work, but am feeling like I need a help of a good histopathologic can you help me with that?
@damienharkin 6 ай бұрын
Hi Sana, I’m not a pathologist, but can provide advice on histological staining techniques to client level members of my KZbin channel.
@soptodipasormi8597 6 ай бұрын
This block is so good.. How to make this block??
@giftysowen1743 7 ай бұрын
I try not to be greedy but I need to make many cross sections of flower which takes about 6 hours and I sometimes loose most parts of it
@damienharkin 7 ай бұрын
We’ve all done this at some point but then realise that patience is essential.
@ivanakancheva5775 8 ай бұрын
Could someone please advice on how to position the knife so that is cuts the tissue well?i struggle with positioning the knife which was not touched upon much? Really nice and clear video, thank you a lot!! :)
@damienharkin 8 ай бұрын
Hi Ivana, There are multiple factors to consider here starting with the specimen itself. During embedding, the tissue should be oriented such that it display least resistance when passing the blade. For example, for skin, cut from dermal side first and epidermis last. The angle of the block face should match the blade and there is usually a recommended angle mark on the blade holder. The knife holder can usually be moved sideways to enable a fresh area of blade to be used when cutting best quality sections. You should also cool your block if excessive crumpling occurs. I hope that helps a little. 🤓
@TheModernVictorian 8 ай бұрын
Thank you very much for your videos. I have found myself interested in microscopy and making slides so I will eventually probably try a few staining approaches when my microtome arrives. I wondered if you had any knowledge of inkjet dyes? When I did a preliminary search I found that they do have the crazy kind of molucular structure as some of the obvious staining dyes, and they certainly stain my hands for a few days after refilling my printer. I am going to test them out once I know that I can perform a simple staining process from end to end with correct stains, then I will try and replace real stains with inkjet dye inks and see how they fair up, but I wondered if you had any opinions on this? Copper Phthalocyanine is one example of a common ink used for cyan channels on a printer.
@damienharkin 8 ай бұрын
I don’t have experience using inkjet dyes, except that copper pthalocyanine is used widely in histology to stain acidic mucosubstances. It is known as Alcian blue in histology.
@astherfe 8 ай бұрын
As an anatomic pathologist. Much appreciation for this video! Hope you can sample other tissue such as lymph node.
@damienharkin 8 ай бұрын
Many thanks for the positive feedback. Since we’re a teaching laboratory we don’t often get access to fresh lymph nodes but this is a great idea for future videos.
@miftajannah6328 8 ай бұрын
hi can i ask? first, thankyou so much u making this video. it helpss. much. when we incubate the working silver methanamine solution. can I drip that solution in to section and incubate in 65 degree celcius on hybridizer/ hot plate for 30-60 minutes?
@damienharkin 8 ай бұрын
Hi, this sounds like an interesting idea and is worth trying, however would suggest covering the slides with a lid to better maintain an even temperature and to reduce drying of solution at the edges. You could also try preheating the coplin with methenamine silver in a water bath. There will be more than one way to achieve a satisfactory result but microwave oven has always worked well for me.
@miftajannah6328 8 ай бұрын
@@damienharkin ah thankyou for the insight. i will try to preheat the reagent first and do drip the solution to slide inside the hybridizer with lid , its about reagent consumption matter for my customer. 😁🙏
@Viralvangaad 8 ай бұрын
So detailed, Thank you
@damienharkin 8 ай бұрын
You’re welcome 😊
@Thana_m 9 ай бұрын
How do you go back if you have over-differentiated the elastin fibre as seen in the video
@damienharkin 9 ай бұрын
Just reapply the Verhoeff’s Hx solution again for a further 10 minutes.
@Thana_m 9 ай бұрын
@@damienharkin and there is no need to differentiate again in ferric chloride or re-stain with van Gieson’s right?
@damienharkin 9 ай бұрын
If you stain with Hx then you may need to redo the Ferric chloride, then do the VG last when happy with the Hx@@Thana_m
@moksy911 9 ай бұрын
Thanks for this video. Why do we need to bake the slides in the oven after mounting?
@damienharkin 9 ай бұрын
You need to bake in order to ensure good attachment of sections to the slide.
@christopherchong3899 9 ай бұрын
Appreciated this advice about cooling the block - 3 years into a PhD project and the tip had never come up *sigh* - hopefully improved tissue sections to come.
@christopherchong3899 9 ай бұрын
Btw safety first! Should definitely have those gloves on around the microtome blade 🔪
@damienharkin 9 ай бұрын
Thanks Christopher! Good luck with the PhD!
@damienharkin 9 ай бұрын
Thanks for raising this point. It’s been raised before and I’ve yet to really respond properly. The microtome blades are certainly very sharp, and so every care should be taken. The safety measures that we employ are generally sufficient to reduce the risk of a cut. Wearing latex or nitrile gloves may well reduce risk further, but the experienced microtomists that I know, don’t wear them. The tissue itself should be safe as has been fixed in formaldehyde and embedded in paraffin, but we do recommend wearing gloves if sectioning brain or spinal cord since prions (e.g. CJD or vCJD), if present, remain infectious.
@itziris8345 9 ай бұрын
As a laboratory student when i found your videos it meant the world to me they are sooo sooo soo helpfuul thank u soo much for the efforts ❤❤❤
@damienharkin 9 ай бұрын
The channel is made for people like you so I’m glad that you’ve found it useful. Please share your experience with others so that I can grow the channel.
@felipedeandradevieiraalves2265 9 ай бұрын
Hello Damien, thanks for this video, very informative. May i ask if you know an alternative option for the martius yellow dye?
@damienharkin 9 ай бұрын
Interesting challenge. Tartrazine, picric acid and Saffron are all yellow dyes, BUT you would have to ensure that they are retained within the RBC throughout all the remaining staining steps. They may well be lost due to differences in molecular weight and solubility compared to Martius yellow. Have a go and let me know how it turns out.
@felipedeandradevieiraalves2265 9 ай бұрын
@@damienharkin Thanks for the reply. I'll try it soon!
@Sabrina-ch1rt 10 ай бұрын
Hello Damien, I'm having issues with our MSB staining specifically on bone marrow trephines. Our staining protocol is similar but we don't post fix, and to be honest I'd never heard of that! Do you think the post fixation is a possible cause of our poor MSBs, do you have any other tips for MSB staining on BMT specifically? Many thanks.
@damienharkin 10 ай бұрын
Hi Sabrina, post-fixation can make a huge difference if initial fixation is incomplete. Are you able to send me a few pictures?
@Sabrina-ch1rt 9 ай бұрын
@@damienharkin Hello, thank you for the reply. Yes I will do when I'm back at work on Wednesday. Thanks again!
@19turan 10 ай бұрын
You are awesome! Thank you very much for such clear explanation!
@damienharkin 10 ай бұрын
You're very welcome!
@hannahajuri6243 10 ай бұрын
As a medical lab student who was lost.. Wanna say thank you lots ❤❤
@damienharkin 10 ай бұрын
You are most welcome Hannah. Let me know if there are any other topics that you would like to see covered. The online course is also still available for Trainee Histologist level members and above.
@lotharmayring6063 10 ай бұрын
Wieso muss denn beim Koehlern der Kondensor zentriert werden. Seine optisch Achse sollte doch in jedem Fall mit der optischen Achse des Objektives uebereinstimmen und nicht mehr veraendert werden. Wohl aber sollte die Leuchtfeldblende zentrierbar sein. Das bedingt auch immer eine zentrierbare Punktlichtquelle.
@damienharkin 10 ай бұрын
A centred light source enables more even illumination of the area of interest. Since the condenser lens focuses light from the light source, then it should be centred. Closing the field iris diaphragm provides an approximate focal point to achieve this.
@lotharmayring6063 10 ай бұрын
@@damienharkin first the light source has to be centered to his iris. Diffusing filters can make the source more even. The condenssor iris should be in the middle of the picture centering it. The condensor iris should be als closed as possible , because we all will prefer interference microscopy. At least the lightfield-iris can be centered to so called Keohler-iris. But in many cases it gives a better image using oblique DIC-like illumination. Koehler-illumination is only usefull for the factory aligning and testing their optics not for good microscopers
@damienharkin 10 ай бұрын
Thanks for the feedback.
@BritishBeachcomber 10 ай бұрын
Great video. I've just upgraded to a similar microscope so I'll be running through the procedure soon. Just one thing. After each adjustment it would be useful if you could show the "before" image alongside, so we can see the improvement.
@damienharkin 10 ай бұрын
Yes, good idea. I thought about doing this but wasn’t sure that the difference would be as obvious when viewed on KZbin. I do notice a discernible difference by eye when looking down the scope. Let me know how you go.
@TouibaMayar 10 ай бұрын
what is programe name?
@damienharkin 10 ай бұрын
The video displays Adobe Photoshop.
@linhmaithuy6531 11 ай бұрын
Have a great day sir. I have a question: with spleen, lymph node specimens, should the section after being embedded in paraffin be cut thinner or thicker than usual ? And why we shoud do that ? Thanks for answering me !
@damienharkin 10 ай бұрын
I think this depends on a number of things. Lymphoid tissue is generally more fragile owing to less ECM components and higher density of cells, so you may gain advantage by cutting slightly thicker sections, but generally this issue can be addressed via use of picric acid in fixative solutions such as Bouin’s.
@linhmaithuy6531 10 ай бұрын
@@damienharkin yeah i got it. Thank u so much.
@linhmaithuy6531 11 ай бұрын
Sir I have a question about bone calcification: For unfixed bones, which of the following solutions should be used: formalin - nitric acid solution or formalin - formic acid solution? Please help me, thanks a lot.
@damienharkin 11 ай бұрын
Hi Linh, Nitric acid is a stronger acid and so decalcifies bone more quickly, but it is also more damaging. So if starting with non-fixed bone, I would suggest combining the formalin with the weaker and therefore more gentle formic acid.
@EclipsegrendaTv Жыл бұрын
This is useful to me, i have an examination tomorrow at Mbarara university in uganda❤, thenks
@damienharkin Жыл бұрын
You are most welcome. Good luck with the exam!
@katyng29 Жыл бұрын
Thank you very much, your explanation is very well!!!
@damienharkin Жыл бұрын
You are welcome!
@suzanelzeyn Жыл бұрын
Interesting 👌...I have a question..what is the type of camera you are using for the point of view camera view? I could not find a very good quality camera resolution that can record videos. Thank you so much for your videos🙌
@damienharkin Жыл бұрын
I use a GoPro camera mounted using a head strap.
@nonysweet7506 Жыл бұрын
Thank you so much 🙏🏼
@damienharkin Жыл бұрын
You’re welcome 😊
@samarsliman4561 Жыл бұрын
Good morning professor my slid didn't take hematoxylin i leave inside it for 15 minute 😢why? This is how i do staining Oven 90 for 1 hours 3 xyline each 15 minute 3 Alcohol each 10 minute Water 10 minute Hematoxylin 15 minute Water Eosin 3 alcohol each 1 minute Pleas please help me😢 Any one see my comment pleas help
@damienharkin Жыл бұрын
Hello. 15 minutes should certainly be long enough to see some staining with Hx. Are you preparing the Hx yourself or buying from a supplier. Assuming that the Hx Solution is fine, it is possible to remove if treat for too long in acid alcohol (ie. if using a regressive staining technique), but you don’t make mention of using this. Another possibility is that slides have not been dewaxed sufficiently to allow entry of stains, but this seems unlikely given your time in xylene. Does the eosin stain the tissue at all?
@damienharkin Жыл бұрын
Another possibility is that you are heating your slides at 90 degrees rather than 60-70 degrees C. This should be ok, but just something to consider in case causing some damage to sections. I also assume that the tissue has been initially fixed and processed correctly.
@19turan Жыл бұрын
Would be able to demonstrate Oil Red O stain? Thank you in advance
@damienharkin Жыл бұрын
Great suggestion!
@katyng29 Жыл бұрын
Good video which help me to improve my staining skill. ❤
@damienharkin Жыл бұрын
Glad it was helpful!
@kayemimages395 Жыл бұрын
Excellent. Good to see a demo with "warts and all"
@damienharkin Жыл бұрын
Glad you liked it!
@georgechandler4664 Ай бұрын
Yes, the "warts" were the most informative parts!
@fadyaaqilah Жыл бұрын
excuse me, I want to ask something. If we want to observe the histopathology of specific inflammatory cells such as macrophages, neutrophils, and lymphocytes in mouse skin tissue, is it enough to just use HE staining? or should use immunohistochemical staining?
@damienharkin Жыл бұрын
Good question. While the basic morphological characteristics of different leukocytes can often be seen quite well in H&E stained sections (e.g., polymorphonuclear leukocytes versus monocytes and lymphocytes), immunohistochemistry enables specific subsets of leukocytes to be demonstrated (e.g., CD4 versus CD8 positive lymphocytes).