This is a super cool video! I am interested in whole mouth in situ hybridisation and currently isolating H4 gene for my plant of interest. I've been doing some research about how different ISH and WMISH are from each other and I didn't understand what are the differences in practical level. I would be so happy if you can give me a hand!
Hi Molly what lab software were you using for the digital Cyto? I work for a Cyto company that captures slides from a scope at cytology and pathology type slides. Learning more about FISH as I have to do a presentation on Monday. Thanks for making biology fun!
We use a Zeiss Axioimager & their Zen Blue software - we often counterstain our in situs with calcofluor white to get better contrast on our cell walls, so in that case we overlay the brightfield image (to visualize the probe) with the fluorescent image that detects the calcofluor (and helps the cells that don't have probe show up better in the final image)
It's uncanny how identical your lab looks to my old lab when I was at the University of Missouri! And that was some mighty fine in situ work you did there.
This topic is not under my expertise. But because the several solutions, it seems this kind of experiment is very laborious. Congratulations for the scientific efforts!
You are absolutely right, it is an *intensely* laborious 2-3 days-long experiment! Very disheartening when it doesn't work because you have to start from scratch, but SO satisfying when you finally get results!
Can you please do a video on the design process of this experiment? How did you design the rna probe and antibody? Is there a simple online tool where I can design a tool and have a company manufacture it for me?
Great idea, I would love to make a video about this one day! In the meantime, here is some info: Luckily you don't need to design the antibody - we use the commonly available anti-DIG antibody, so you just need to incorporate DIG-labeled NTPs into your probe during transcription. You can amplify your probe fragment from cDNA - you just want to make sure it's in a specific region of your gene of interest so that you don't detect closely related genes. We usually design our probes with one end in the UTR to ensure specificity, but definitely do an alignment with other members of the gene family to check where the conserved regions are. Here is our lab's protocol - this is a slightly older version, again I just amplify from cDNA and use that fragment directly in the topo reaction. There might be a service that would make probes for you but I imagine it would be very pricey! kramerlab.oeb.harvard.edu/files/kramerlab/files/in_situ_protocol_corrected-2.pdf?m=1430323911
@@ScienceIRL also did you use crispr to turn off the gene you suspected controlled petal curvature? That would just be changing the start codon of your gene of interest correct?
@@milesgiehtbrock8510 we don't have stable transgenics in Aquilegia yet so I can only use VIGS (Virus induced gene silencing) to target genes of interest, unfortunately. For VIGS we usually use the same target fragment as our in situ hybridizations.
This video is my introduction to the channel and i must say, i appreciate your efforts here. All in all, well performed & explained!
So interesting! I have never seen a scientific video so vividly like this!
Cool same technique used to look at tissue or organ biopsies
So I've heard in situ pronounced "in sit chew", "in see 2" and "in sit 2". What say you?
Best explanation ever :)
Thank you very much for such a nice and interesting video for such hectic technique ;)))
This is a super cool video! I am interested in whole mouth in situ hybridisation and currently isolating H4 gene for my plant of interest. I've been doing some research about how different ISH and WMISH are from each other and I didn't understand what are the differences in practical level. I would be so happy if you can give me a hand!
Her supervisor seems so proud of her.
Great episode! Thanks for teaching us about in situ hybridization! So fun to see all the steps instead of just an abstract explanation!
Hi Molly what lab software were you using for the digital Cyto? I work for a Cyto company that captures slides from a scope at cytology and pathology type slides. Learning more about FISH as I have to do a presentation on Monday. Thanks for making biology fun!
We use a Zeiss Axioimager & their Zen Blue software - we often counterstain our in situs with calcofluor white to get better contrast on our cell walls, so in that case we overlay the brightfield image (to visualize the probe) with the fluorescent image that detects the calcofluor (and helps the cells that don't have probe show up better in the final image)
Great video. Very helpful.
bro...thank you so much
Hi! I was wondering if you had a copy of the protocol you used. Thx!
Here you go! kramerlab.oeb.harvard.edu/files/kramerlab/files/in_situ_protocol_corrected-2.pdf?m=1430323911
It's uncanny how identical your lab looks to my old lab when I was at the University of Missouri! And that was some mighty fine in situ work you did there.
Ah lab deja vu, I know it well! And, thank you :) If only there were an IRL time-lapse button for all those solution changes!
This topic is not under my expertise. But because the several solutions, it seems this kind of experiment is very laborious. Congratulations for the scientific efforts!
You are absolutely right, it is an *intensely* laborious 2-3 days-long experiment! Very disheartening when it doesn't work because you have to start from scratch, but SO satisfying when you finally get results!
@@ScienceIRL Perfect!😊😀
Very interesting. Thank you!
Love your videos and you
Well done 👍
Can you please do a video on the design process of this experiment? How did you design the rna probe and antibody? Is there a simple online tool where I can design a tool and have a company manufacture it for me?
Great idea, I would love to make a video about this one day! In the meantime, here is some info:
Luckily you don't need to design the antibody - we use the commonly available anti-DIG antibody, so you just need to incorporate DIG-labeled NTPs into your probe during transcription. You can amplify your probe fragment from cDNA - you just want to make sure it's in a specific region of your gene of interest so that you don't detect closely related genes. We usually design our probes with one end in the UTR to ensure specificity, but definitely do an alignment with other members of the gene family to check where the conserved regions are. Here is our lab's protocol - this is a slightly older version, again I just amplify from cDNA and use that fragment directly in the topo reaction. There might be a service that would make probes for you but I imagine it would be very pricey! kramerlab.oeb.harvard.edu/files/kramerlab/files/in_situ_protocol_corrected-2.pdf?m=1430323911
@@ScienceIRL also did you use crispr to turn off the gene you suspected controlled petal curvature? That would just be changing the start codon of your gene of interest correct?
@@milesgiehtbrock8510 we don't have stable transgenics in Aquilegia yet so I can only use VIGS (Virus induced gene silencing) to target genes of interest, unfortunately. For VIGS we usually use the same target fragment as our in situ hybridizations.
@@ScienceIRL Thanks! Just learned something new. I Will research that now.
Wonderful Episode!
:)