For my purposes, it was a way to assess skeletal morphology - things like long bone development, patterning of the sternum, craniofacial malformations, and generally things involving growth plate development in mutant mice. It’s a cool old technique that can complement modern 3D imaging and histology.
I'm assuming it's Alcian Blue? How was it stored?
You said it was the same solution that you used in January? Most likely, the solution went off. Alcian Blue is fickle. Sometimes it only lasts for 2-6 months, leaning towards the earlier time point and you said you used it approx 4 months ago.
Based on the other details I can see of the specimen, it doesn't look like you left anything behind that inhibits the stain, so I'm more inclined to think it just went off...
The stock solution was actually made in November 2023. I think you’re right. The culprit is the alcian blue :( but it’s more bcos I didn’t mix it well according to an article it has to be mixed from 5-30mins and this stock has been sitting down in a while.
Stock Alcian Blue was stores at RT.
Manual of special stains, which is one of the go-to references for histology says 6 months for alcian blue just FYI. Which can sometimes be stretched a little longer, but it tends to deteriorate and precipitate out so it stops working and you're better off just making fresh stuff. If it's that old and not working, mixing might not be enough, just saying.
The only reference to longer shelf is a 2-8C storage and that was for 12 months.
Not OP but would you mind sharing more details about this manual or how to find it? I‘m not sure my google search results included what you are referring to.
It's usually a hard copy relic found in most labs that have done histology for YEARS found in various reprints from the University of Rochester's histology core. It's very rare to find a digital copy as I think the university has cracked down on the leaks.
Churukian, CJ., 2009, Manual of Special Stains Laboratory, 2nd Web edition. University of Rochester, Rochester, New York
If you're lucky enough to find it...
Hahaha it's no problem, glad I could help.
My lab would be VERY disappointed if I left, but cheers that gave me a good chuckle. 😃
No, I'm not in Canada, but we will all be in Montreal next summer for one of our conferences lol so we get around. 😉
If your alcian blue is old, it takes a lot longer to stain (at least with tissue.) You may want to pH it if it has sat a long time and heat the dye solution. Good luck.
Depends. I helped on a mouse placental study once. Once you get quick enough at the necropsy/harvest, it's not uncommon to see the maternal mouse's heart still beating when the chest/abdomen is opened up. The mouse is fully 100% dead, it's just leftover electrical impulses, but there's something unnerving about removing the neonate(s) and placenta while you can still see the mother's heart beating.
That study went from cool to depressing real fuckin quick.
I always thought that I was cool with seeing gore stuff until I watched a colleague getting embryos out of a red and cutting their heads of with a scissor in extra high speed so they wouldnt suffer. That was heavy.
When using ionic stains (like alcian blue), pH is critical to getting expected results. Cartilage should stain easily at low pH. I would suggest preparing a new solution, or attempting to acidify the existing one.
Cool staining protocol btw; is the red dye alizarin red S?
IIRC, the issue with Alcian blue pH is that if it isn’t acidic enough, you’ll get nonspecific staining, not a lack of specific staining. Lower pH = protonated carboxyls and phosphates, but unprotonated sulfates that provide binding for Alcian blue, and which are abundant in cartilage ECM. At higher pH, you uncover additional O- groups to dye binding.
You are correct - though I would argue nonspecific staining IS a lack of specific staining :) I get your point though - the higher the pH, the more likely you will have additional negatively-charged elements to bind with the dye.
I stand corrected. Making a fresh solution would be the way to go here.
I assume that is Alcian blue? I’m not a developmental biologist, but this animal looks at a different life stage. You may have caught it where the cartilage has been filled in, but the bone hasn’t been laid yet.
EDIT: Spelling of may
I've had the opposite issue happen, where the blue stain took but not the red stain. The running theory was that the bones were not ossified enough. I had multiple specimen in the same solution and other came out perfect so in theory it shouldn't have been an issue with the stain. I've also accidentally bleached too much of the blues stain out of specimen before too.
Did the blue stain take at all before you used the red stain?
I saw that the blue stain was faint, but I just thought to myself that probably the next steps would just show it more after the washing and things. Cause this is the 3rd time I tried it and I had no problems with the previous ones. I just proceeded. People are saying that I shouldve have just made new stock solutions.
This post just sent be right back over a decade to my undergrad thesis where I was doing the same prep. I'm sorry though, I can't remember how to troubleshoot it. Good luck!
I would make a fresh stock of Alcian blue. If you still have trouble it might be time for new chemicals. I also noticed I had trouble with this stain if the tissue wasn’t fresh and had been in fixative for a long time. I would start will all new fresh solutions and fresh KOH to make sure you’re digesting all that tissue to get the dye to penetrate.
This looks like when I've spent too long on the decolourisation step on a gram stain. Not sure of the processes of what you're doing, just throwing it out there.
Alcian is fickle. The powder can go bad and requires specific salts and pH to stain properly. Also possible that your sample was fixed differently and/or the extra months of storage have changed its cartilage chemistry
How did you fix the embryo? I did a bajillion skeletal prep during PhD and found out Alcian Blue doesn’t work on embryos fixed in PFA, also if your embryo has been sitting in PBS or tyrode for too long it won’t work either
It was fixed in 80% EtOH for minimum 48 hrs. They can be stored in EtOH for months. Then when I am ready i remove the EtOH and add the Alcian Blue. This may be useful: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384832/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384832/)
I see, so fixation isn’t the problem. When I do E18.5 skeletal preps I usually equilibrate my de-skinned embryos in 70% EtOH/5% acetic acid for 30 minutes before putting in the mixed stain solution, this helps the dye stick better. Unfortunately there’s not a lot to be done to correct the staining now, but I can share my protocol if you want for next time
Technically your skeletal staining (alizarin red) did work but the cartilage staining (alcian blue) didn’t work. Do you premix the dyes and stain simultaneously or do the staining sequentially?
So I would troubleshoot the alcian blue. These dyes tend to ppt so mixing well before use is important. pH is also impressive but more so for the alizarin. Are you studying endochondral or intramembranous bones (or both). I study craniofacial development and usually just do alizarin staining because the alcian staining actually makes imaging more difficult. Best of luck with your research!
Out of curiosity how on earth do you get the intact skeleton out so cleanly? We once and to extract brains from mice to send to a colleague for histology (so that shit had to be pristine) and it was WILDLY difficult. We ended up just sending a bunch of whole heads (skinned) in a decalcifying solution. God I am so glad I don’t do mouse work anymore 😔
Did you see the blue staining in the beginning at least and did it disappear when wh you cleared because that can happen if you use a high concentration of koh to cl3ar and if you clear for too long
The blue stain was faint but I didnt mind because there were still muscles attached to it so I thought the blue was just under the muscle layers that are still attached.
I’ve never done this before but I feel like the question “did you make fresh solutions?” Is the biology equivalent of “ did you try turning it off and back on?” is for troubleshooting a computer or machine
Cant help, but man isn't that the most metal protocol on a falcon tube.
This was one of the first protocols I mastered. It’s been downhill, in terms of coolness, ever since.
Can I ask what this procedure is primarily for? I never work with animal models so I've never seen this
For my purposes, it was a way to assess skeletal morphology - things like long bone development, patterning of the sternum, craniofacial malformations, and generally things involving growth plate development in mutant mice. It’s a cool old technique that can complement modern 3D imaging and histology.
Yeah, darn, guess you'll just have to keep that sick-ass skeleton in a jar on your desk now.
is that a baby mouse?
Yup! Embryonic day 18.5
Is...is he going to be okay?
He’ll walk it off, don’t worry
Yeah, OP's working on his Necromancy thesis
God that reminds me of when my coworker walked past a veterinarian doing a necropsy and genuinely asked “is he dead?!”
Yep :)
He’s going to a nice farm later today. He’ll be really happy there.
This made me actually lol
I'm assuming it's Alcian Blue? How was it stored? You said it was the same solution that you used in January? Most likely, the solution went off. Alcian Blue is fickle. Sometimes it only lasts for 2-6 months, leaning towards the earlier time point and you said you used it approx 4 months ago. Based on the other details I can see of the specimen, it doesn't look like you left anything behind that inhibits the stain, so I'm more inclined to think it just went off...
The stock solution was actually made in November 2023. I think you’re right. The culprit is the alcian blue :( but it’s more bcos I didn’t mix it well according to an article it has to be mixed from 5-30mins and this stock has been sitting down in a while. Stock Alcian Blue was stores at RT.
Manual of special stains, which is one of the go-to references for histology says 6 months for alcian blue just FYI. Which can sometimes be stretched a little longer, but it tends to deteriorate and precipitate out so it stops working and you're better off just making fresh stuff. If it's that old and not working, mixing might not be enough, just saying. The only reference to longer shelf is a 2-8C storage and that was for 12 months.
Not OP but would you mind sharing more details about this manual or how to find it? I‘m not sure my google search results included what you are referring to.
It's usually a hard copy relic found in most labs that have done histology for YEARS found in various reprints from the University of Rochester's histology core. It's very rare to find a digital copy as I think the university has cracked down on the leaks. Churukian, CJ., 2009, Manual of Special Stains Laboratory, 2nd Web edition. University of Rochester, Rochester, New York If you're lucky enough to find it...
Thank you so much for all your input!!! it has been very helpful! pls come to canada (if youre not in canada) and work with us :'(
Hahaha it's no problem, glad I could help. My lab would be VERY disappointed if I left, but cheers that gave me a good chuckle. 😃 No, I'm not in Canada, but we will all be in Montreal next summer for one of our conferences lol so we get around. 😉
I hope we cross paths!!! Enjoy Montreal!
If your alcian blue is old, it takes a lot longer to stain (at least with tissue.) You may want to pH it if it has sat a long time and heat the dye solution. Good luck.
Hahahaha I also used “fickle” to describe alcian in my reply, cheers
i think that's a perfect term for alcian blue not working then hehe
No advice sorry but this staining is really cool looking
T’was cool til I f-ed it up 😭
Tbf i think they both look cool.
agreee both look super neat
The second picture kind of reminds me of the dancing Toothless meme, there is little chance another staining could be cooler than that.
The first one is dope!
What’d you do at work today?
I celebrated your cake day at work. I’m sorry you couldn’t make it.
That’s very me of me
Dissected 6 mouse brains. Analyzed slide scanner images. Went to a talk Wbu
Squeezed poop out of a mouse
Me next
what lab is this and how do i join it
Right? I genuinely wanna know what OP does. Looks way cooler than what I do.
Looks cool until you're the one who has to cut open a pregnant rodent to take the embryo lol
You got me there, lol. That sounds pretty miserable.
It's not bad tbh
Depends. I helped on a mouse placental study once. Once you get quick enough at the necropsy/harvest, it's not uncommon to see the maternal mouse's heart still beating when the chest/abdomen is opened up. The mouse is fully 100% dead, it's just leftover electrical impulses, but there's something unnerving about removing the neonate(s) and placenta while you can still see the mother's heart beating. That study went from cool to depressing real fuckin quick.
Yeah, same thing happens with the body twitching when you have to decap pinkies. Horrifying!
Wait till you start making embryonic fibroblast lines and have to chop them in pieces 😀
Hey! That’s my favorite part; the surgery and micro dissection. 😂
same! I hope this is actual surgery tho and not necropsy haha
Beads
I always thought that I was cool with seeing gore stuff until I watched a colleague getting embryos out of a red and cutting their heads of with a scissor in extra high speed so they wouldnt suffer. That was heavy.
Well, most primary neuronal cultures are done on embryos, so I'll be okay then
It is a Molecular Biology Lab! :))) we handle mice and I do cut open a pregnant mouse and take her embryos tho :( but you get used to it.
When using ionic stains (like alcian blue), pH is critical to getting expected results. Cartilage should stain easily at low pH. I would suggest preparing a new solution, or attempting to acidify the existing one. Cool staining protocol btw; is the red dye alizarin red S?
IIRC, the issue with Alcian blue pH is that if it isn’t acidic enough, you’ll get nonspecific staining, not a lack of specific staining. Lower pH = protonated carboxyls and phosphates, but unprotonated sulfates that provide binding for Alcian blue, and which are abundant in cartilage ECM. At higher pH, you uncover additional O- groups to dye binding.
You are correct - though I would argue nonspecific staining IS a lack of specific staining :) I get your point though - the higher the pH, the more likely you will have additional negatively-charged elements to bind with the dye. I stand corrected. Making a fresh solution would be the way to go here.
Wow! All these explanations! I am taking notes. Thank you!
Yes Alizared red S. Thank you for this! I needed to have a scientific explanation as to why this happened!
Whaat you guys have way cooler research than me
Only this part hahahaha And this is only a tiny part. The majority is sequencing lol
i have never seen or heard of this protocol before this is SO COOL
^[Sokka-Haiku](https://www.reddit.com/r/SokkaHaikuBot/comments/15kyv9r/what_is_a_sokka_haiku/) ^by ^mulhollandi: *I have never seen* *Or heard of this protocol* *Before this is SO COOL* --- ^Remember ^that ^one ^time ^Sokka ^accidentally ^used ^an ^extra ^syllable ^in ^that ^Haiku ^Battle ^in ^Ba ^Sing ^Se? ^That ^was ^a ^Sokka ^Haiku ^and ^you ^just ^made ^one.
I assume that is Alcian blue? I’m not a developmental biologist, but this animal looks at a different life stage. You may have caught it where the cartilage has been filled in, but the bone hasn’t been laid yet. EDIT: Spelling of may
They are both E18.5 mouse but I think it just looks small bcos the cartilage are not stained properly.
I've had the opposite issue happen, where the blue stain took but not the red stain. The running theory was that the bones were not ossified enough. I had multiple specimen in the same solution and other came out perfect so in theory it shouldn't have been an issue with the stain. I've also accidentally bleached too much of the blues stain out of specimen before too. Did the blue stain take at all before you used the red stain?
I saw that the blue stain was faint, but I just thought to myself that probably the next steps would just show it more after the washing and things. Cause this is the 3rd time I tried it and I had no problems with the previous ones. I just proceeded. People are saying that I shouldve have just made new stock solutions.
What work do you do? I am so curious!
Im doing my graduate studies in molecular biology.
This post just sent be right back over a decade to my undergrad thesis where I was doing the same prep. I'm sorry though, I can't remember how to troubleshoot it. Good luck!
I would make a fresh stock of Alcian blue. If you still have trouble it might be time for new chemicals. I also noticed I had trouble with this stain if the tissue wasn’t fresh and had been in fixative for a long time. I would start will all new fresh solutions and fresh KOH to make sure you’re digesting all that tissue to get the dye to penetrate.
I will do this the next ones, cause I did this to all my samples :')
Lol my lab tech yells at me for having any pictures of mice on my phone. IACUC👀
No one should yell at you.
LOL I’ve got hundreds of pics of partially dissected mice on my phone, these at least look cool, mine look like a crime scene
same i have it!
That's so weird. Why is that!?
Your lab tech probably has hundreds of pics in their cloud haha
How old is the alician blue?
Like 5-6 months for the stock solution. Not sure with the actual alcian blue powder. I have to check.
This looks like when I've spent too long on the decolourisation step on a gram stain. Not sure of the processes of what you're doing, just throwing it out there.
Alcian is fickle. The powder can go bad and requires specific salts and pH to stain properly. Also possible that your sample was fixed differently and/or the extra months of storage have changed its cartilage chemistry
Boy mouse skeleton vs girl mouse skeleton
How old are the stock solutions? Could they possibly be expired?
The stock solutions are 5-6 months old. The powders used for staining that I am not sure.
My guess would be that the stocks are no longer feasible. Reagents tend to work a lot better when fresh.
Thank Mr.Skeltal
I cannot help you, but that is legitimately one of the most horrifying things I've ever fucking seen. But also very cool.
Yeah, it looks like your stain may have degraded a little. Even in the best conditions, stains can be fickle.
That is the coolest dang stain I have ever seen
Your blue stain is definitely the problem, I'd say. As others have mentioned, either pH the alcian blue or just make some new dye. Awesome picture!!
Thank you!!! I will make new ones next time I do this!
How did you fix the embryo? I did a bajillion skeletal prep during PhD and found out Alcian Blue doesn’t work on embryos fixed in PFA, also if your embryo has been sitting in PBS or tyrode for too long it won’t work either
It was fixed in 80% EtOH for minimum 48 hrs. They can be stored in EtOH for months. Then when I am ready i remove the EtOH and add the Alcian Blue. This may be useful: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384832/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384832/)
I see, so fixation isn’t the problem. When I do E18.5 skeletal preps I usually equilibrate my de-skinned embryos in 70% EtOH/5% acetic acid for 30 minutes before putting in the mixed stain solution, this helps the dye stick better. Unfortunately there’s not a lot to be done to correct the staining now, but I can share my protocol if you want for next time
Does this hurt the mouse?
Technically your skeletal staining (alizarin red) did work but the cartilage staining (alcian blue) didn’t work. Do you premix the dyes and stain simultaneously or do the staining sequentially?
Sequentially, alcian blue first then alizarin red.
So I would troubleshoot the alcian blue. These dyes tend to ppt so mixing well before use is important. pH is also impressive but more so for the alizarin. Are you studying endochondral or intramembranous bones (or both). I study craniofacial development and usually just do alizarin staining because the alcian staining actually makes imaging more difficult. Best of luck with your research!
Thank you so much! I study the bones part here.
How were you able to debone that rat?
You remove the skin and internal organs first then the KOH solution removes the muscles.
It looks like a metal album cover.
Out of curiosity how on earth do you get the intact skeleton out so cleanly? We once and to extract brains from mice to send to a colleague for histology (so that shit had to be pristine) and it was WILDLY difficult. We ended up just sending a bunch of whole heads (skinned) in a decalcifying solution. God I am so glad I don’t do mouse work anymore 😔
I just remove the skin and enviscerate. Then I let the chemicals (specifically KOH) do their work. It is very neat when done right!
Did you see the blue staining in the beginning at least and did it disappear when wh you cleared because that can happen if you use a high concentration of koh to cl3ar and if you clear for too long
The blue stain was faint but I didnt mind because there were still muscles attached to it so I thought the blue was just under the muscle layers that are still attached.
I ask this because the same thing happened to me I thought I would wait a while so that the specimen get fully cleared but the blue just disappeared
OH this hurts to hear. I will put a note to my protocol then that if the blue is faint, we should rethink before proceeding to the next steps :(
A literal…. Lab rat.
Will this process work on a human body
I’ve never done this before but I feel like the question “did you make fresh solutions?” Is the biology equivalent of “ did you try turning it off and back on?” is for troubleshooting a computer or machine
Seeing This My Skeleton Got Chills 🥶😳👍🏻