The temperature is always difficult on these. It could be your sample is too warm and when you go to cut, the blade just kind of scapes against it but doesn't cut because it is not frozen enough. I used to do developmental studies and cutting at different stages of development of the same tissue changed my cutting temperature from -16C to -35C (differences in extracellular matrix composition). Also, cutting thin sections is really hard and avoid it if you can. My boss wanted 2um sections, and I did it, but it took hours per sample to get the right temperature and cut them.
If you want to try and test it, you could put a plate in the freezer to chill it down. While cooking, add a drop from your pot on that plate and put it in the freezer for like 30 seconds to let it cool so you can check it's consistency.
Well it all depends, the ROCKi helps with the recovery coming out of thaw. But it has more of an impact if your cells were frozen as single cells or as clumps. I used to not use ROCKi when thawing my clump passaged cells, but protocols do use ROCKi to help with viability of clump passaged cells during thaw. For single cells it is definitely important to keep it in ROCKi depending on how dense your cells are.
I usually have good recovery and high enough cell density the next day after thawing that I passage to prepare my cell experiments. But it really is line dependent and how you freeze them down and how well they normally recover.
In many of my slides the fluorescence extremly differs, meaning there are brighter and darker regions. I am very careful when it comes to washing, mixing etc. so I have no idea what is causing this. In general cells that are in "colonies" have a darker fluorescence than those cells that are alone or in small groups.
How long was the incubation for primary and secondary for and at what temperature? Also how are you incubation with enough volume? Are you just submersing your primary/secondary in liquid or drain l sealing it in with your sample?
I pipette 100 µL of the diluted antibody onto parafilm and then place the cover slip on top of this (to not waste antibody). The parafilm is inside a petri dish with a wet kimwipe, so everything stays humid. The primary antibody is incubated over night at 4 °C and the secondary antibody for 2 h at RT in the dark.
I used to always do the same with the parafilm and staining procedure, 100ul of antibody and wet Kim wipe and all. So I do not think it is the staining protocol. Though as some others were stating maybe your fixation. The methanol should be ice cold, and do you rehydrate afterwards for a bit of time? If you want to try and switch to using PFA, you can dilute it in PHEM or Brinkley buffer. See some protocols from Ted Salmon's or Tim Mitchison's labs, I know they may be old protocols, but they have worked well for me for many years.
I commented below that on Zeiss (I have 4 Observer Z1s), that there is a slot below the filter cubes that you can put a slider into. If something is in there and out of place then this can happen. Another question is of you switch the optical path to loom at it with your eyes through the binoculars, do you see the same thing. Lastly, does the objective seem off? The motorized switching can messed up if something spills in there.
I checked the sliders and that didn’t change anything :/ I do see this on the eyepieces too. The objective does look off, i can see that the illumination is not centered over the objective
Well, next door to Owen's is a sushi place called Kusshi, but they have a bar area and are open late (I think 12:30 or 1am during the week). Check out Gilly's nearby if you ever get the chance as they have a good rotating selection on draft and good food, but unfortunately they close at 10pm.
This could still be an effect from the ROCKi after washing out. Though since you are doing clump passaging, you don't need the ROCKi. I only use it when bringing cells out of thaw or doing single cell passaging.
Yeah I've found papers where it's fine as well. We have a cell line with a GFP tagged protein, used for live cell imaging, gorgeous results. When fixed with PFA though there's just strong green background fluorescence, according to a post doc in my lab
Increased background is common and well-known in aldehyde fixation (worse with glutaraldehyde). I used to do Sodium Borohydride to quench that background. MBS fixation is another method that we use which seems to preserve fluorescent proteins and with great signal to noise.
I was thinking the same when I bought it, but I was pleasantly surprised when I drank it. I was getting it for the bottle, but the whiskey itself was pretty good.
Literally started playing again yesterday after a couple years. First game back and our mid just raged after dying twice in 15 minutes and started just feeding. Called it a night after that and contemplating taking another couple year break.
As someone who has recruited some postbacs at NIH in the past. We are given a database with all the applicants. Much of the recruiting is done around January-February, and people who apply after that are likely to miss that recruitment window as most labs will have recruited by then. Applying early should be fine as you will be in that database. It's more of an issue if you aren't there when people start looking. Part of that is because most postbacs come and leave in the summer and so the recruiting is done a few months before that. You can certainly reach out from now, but I would say to start reaching out by the end of the year closer to when most of the recruiting happens.
I know this is a different level but I was really stoked to try milk bar bakeries! Watched her chefs table and was like really excited about the process and flavors! I got a well rounded sampling from the menu and it tasted like grocery store bakery. Definitely ALL margarine. Buttercream was foul, I hate wasting food but this sticks with me because I tossed it all. Inedible.
I remember trying milk bar a several years ago, never been back since. It was pretty disappointing for me, and that was without knowing about all the hype. Just happened to be walking by after dinner thought I'd try it.
From the blind my favorites from 1st to last were the Filibuster Triple cask, Backbone Uncut 2Bros1Bottle Pick, Davidson Reserve Four Grain, Still Austin & Breckenridge Rum Cask tied (thought they were very different), Maker’s Mark Cape Fear Wood Finish Pick, Kentucky Owl St. Patrick edition & way down dead last was Wilderness Trail BIB Bourbon. The Penelope Rio no one in the group except for me really knew what it was & rated it the highest out of all the bourbons after the blind! But the top four bottles across the board for the blind were Fillistbuter, Still Austin, Backbone & Breckenridge Rum finished. We took notes, wrote what we thought we would pay for each, rated it 1-5 & guessed proof & age.
Apply as a senior. There is no posted cycle, but most of the recruiting is done around January-February for the upcoming summer/fall start. So you should put your application in before then.
I was nearing the end of my deadline for a K99 as a 3rd year postdoc, and my boss after learning that I was focusing on writing my grant was fairly angry at me. He did not want me to spend any time working on it and just focus on doing experiments. He just kept saying everything from that I will never get it, that I need to publish more, to a range of other things. It was at that point I decided that I really did not want to be in academia anymore. I ended up finding a job and then left. He tried to say that if I stayed longer I'd get more papers and get a higher paying job, then it was that I wasn't skilled enough to leave. It was a bit baffling as I left with 4 publications after 4 years (3 first/cofirst author), and 3 out of 4 in high impact factor journals. I think it was just seeing that level of greed to squeeze out as much work out of me while not caring about my future prospects.
I am assuming I do as my post doc also watched me the whole time, but I am definitely going to be more cautious with re-suspending my cells as someone else has also mentioned that! I currently re-suspend the cells with a 1000ul pipette and I usually have about 1-2ml of cells. Would you recommend anything in regards to how I re-suspend the cells?
I think also after you put them in the plate, do you swirl them well. One thing is if a you have a high concentration of cells in the center of the well this can also cause issues as they come together and adhere. I'm just speaking from my experience with iPSCs. Even if you mix before, just handling the plate might make the cells go to the center.
Oh you’re right I put the cells in drops spread around the well and I swirl them after but I do not swirl them directly after I’ve put them in the incubator and that might’ve been the problem! 🥲
Just be aware of that. Give the plate some figure 8s after carrying them to the incubator. Even the movement of carrying them might make them move to the center. I hope it is just a simple fix like this, but I would check the other things as well to be sure.
Thanks for this. It gives me a lot to think about and look into. Redundancy is what we are still discussing how to be done (a division-wide system to interface with as an option). I think the small files is definitely an issue that we were trying to consider the best way to deal with.
I have 6 unopened. 3 are the same bottle that I go through a lot, and 3 are new ones that I want to try. But I have 71 open bottles, half of which are low and will need replacing soon if I can afford lol.
The temperature is always difficult on these. It could be your sample is too warm and when you go to cut, the blade just kind of scapes against it but doesn't cut because it is not frozen enough. I used to do developmental studies and cutting at different stages of development of the same tissue changed my cutting temperature from -16C to -35C (differences in extracellular matrix composition). Also, cutting thin sections is really hard and avoid it if you can. My boss wanted 2um sections, and I did it, but it took hours per sample to get the right temperature and cut them.
It's okay. I found it to be too sweet and fruity for me.
If you want to try and test it, you could put a plate in the freezer to chill it down. While cooking, add a drop from your pot on that plate and put it in the freezer for like 30 seconds to let it cool so you can check it's consistency.
Well it all depends, the ROCKi helps with the recovery coming out of thaw. But it has more of an impact if your cells were frozen as single cells or as clumps. I used to not use ROCKi when thawing my clump passaged cells, but protocols do use ROCKi to help with viability of clump passaged cells during thaw. For single cells it is definitely important to keep it in ROCKi depending on how dense your cells are.
I passaged them as single cells this morning - I eventually decided to just leave them as is.
I usually have good recovery and high enough cell density the next day after thawing that I passage to prepare my cell experiments. But it really is line dependent and how you freeze them down and how well they normally recover.
In many of my slides the fluorescence extremly differs, meaning there are brighter and darker regions. I am very careful when it comes to washing, mixing etc. so I have no idea what is causing this. In general cells that are in "colonies" have a darker fluorescence than those cells that are alone or in small groups.
How long was the incubation for primary and secondary for and at what temperature? Also how are you incubation with enough volume? Are you just submersing your primary/secondary in liquid or drain l sealing it in with your sample?
I pipette 100 µL of the diluted antibody onto parafilm and then place the cover slip on top of this (to not waste antibody). The parafilm is inside a petri dish with a wet kimwipe, so everything stays humid. The primary antibody is incubated over night at 4 °C and the secondary antibody for 2 h at RT in the dark.
I used to always do the same with the parafilm and staining procedure, 100ul of antibody and wet Kim wipe and all. So I do not think it is the staining protocol. Though as some others were stating maybe your fixation. The methanol should be ice cold, and do you rehydrate afterwards for a bit of time? If you want to try and switch to using PFA, you can dilute it in PHEM or Brinkley buffer. See some protocols from Ted Salmon's or Tim Mitchison's labs, I know they may be old protocols, but they have worked well for me for many years.
$70 is okay. Usually I see it $75-85. Based on what you've tried and possibly like, you should like this one. It's nothing crazy with a lower abv.
It's Suffolk, VA. I've seen plenty of crazy stuff there going through that tunnel to Norfolk.
Technically that tunnel is between Portsmouth and Norfolk. Suffolk is about 5-10 miles further up the road.
You are right! It's been too many years since I made that drive.
Sorry - Zeiss Axio Observe Z1
I commented below that on Zeiss (I have 4 Observer Z1s), that there is a slot below the filter cubes that you can put a slider into. If something is in there and out of place then this can happen. Another question is of you switch the optical path to loom at it with your eyes through the binoculars, do you see the same thing. Lastly, does the objective seem off? The motorized switching can messed up if something spills in there.
I checked the sliders and that didn’t change anything :/ I do see this on the eyepieces too. The objective does look off, i can see that the illumination is not centered over the objective
You can try and move the objective into place and see if it clicks into place
Sounds good, but I can't get there until about 10 or so.
Well, next door to Owen's is a sushi place called Kusshi, but they have a bar area and are open late (I think 12:30 or 1am during the week). Check out Gilly's nearby if you ever get the chance as they have a good rotating selection on draft and good food, but unfortunately they close at 10pm.
This could still be an effect from the ROCKi after washing out. Though since you are doing clump passaging, you don't need the ROCKi. I only use it when bringing cells out of thaw or doing single cell passaging.
Yeah I've found papers where it's fine as well. We have a cell line with a GFP tagged protein, used for live cell imaging, gorgeous results. When fixed with PFA though there's just strong green background fluorescence, according to a post doc in my lab
Increased background is common and well-known in aldehyde fixation (worse with glutaraldehyde). I used to do Sodium Borohydride to quench that background. MBS fixation is another method that we use which seems to preserve fluorescent proteins and with great signal to noise.
Definitely a sexy bottle. I’d buy it for the bottle even if the juice inside sucked.
I was thinking the same when I bought it, but I was pleasantly surprised when I drank it. I was getting it for the bottle, but the whiskey itself was pretty good.
I just had my week ruined like this. Hopefully I won't have to go through this again for a while. I definitely drank more this week than usual
Literally started playing again yesterday after a couple years. First game back and our mid just raged after dying twice in 15 minutes and started just feeding. Called it a night after that and contemplating taking another couple year break.
As someone who has recruited some postbacs at NIH in the past. We are given a database with all the applicants. Much of the recruiting is done around January-February, and people who apply after that are likely to miss that recruitment window as most labs will have recruited by then. Applying early should be fine as you will be in that database. It's more of an issue if you aren't there when people start looking. Part of that is because most postbacs come and leave in the summer and so the recruiting is done a few months before that. You can certainly reach out from now, but I would say to start reaching out by the end of the year closer to when most of the recruiting happens.
do you mind if I DM?
Sure
I know this is a different level but I was really stoked to try milk bar bakeries! Watched her chefs table and was like really excited about the process and flavors! I got a well rounded sampling from the menu and it tasted like grocery store bakery. Definitely ALL margarine. Buttercream was foul, I hate wasting food but this sticks with me because I tossed it all. Inedible.
I remember trying milk bar a several years ago, never been back since. It was pretty disappointing for me, and that was without knowing about all the hype. Just happened to be walking by after dinner thought I'd try it.
From the blind my favorites from 1st to last were the Filibuster Triple cask, Backbone Uncut 2Bros1Bottle Pick, Davidson Reserve Four Grain, Still Austin & Breckenridge Rum Cask tied (thought they were very different), Maker’s Mark Cape Fear Wood Finish Pick, Kentucky Owl St. Patrick edition & way down dead last was Wilderness Trail BIB Bourbon. The Penelope Rio no one in the group except for me really knew what it was & rated it the highest out of all the bourbons after the blind! But the top four bottles across the board for the blind were Fillistbuter, Still Austin, Backbone & Breckenridge Rum finished. We took notes, wrote what we thought we would pay for each, rated it 1-5 & guessed proof & age.
I really like all the fillibuster bottles I've had. I've been meaning to go to their distillery next time I am in that area.
Apply as a senior. There is no posted cycle, but most of the recruiting is done around January-February for the upcoming summer/fall start. So you should put your application in before then.
I was nearing the end of my deadline for a K99 as a 3rd year postdoc, and my boss after learning that I was focusing on writing my grant was fairly angry at me. He did not want me to spend any time working on it and just focus on doing experiments. He just kept saying everything from that I will never get it, that I need to publish more, to a range of other things. It was at that point I decided that I really did not want to be in academia anymore. I ended up finding a job and then left. He tried to say that if I stayed longer I'd get more papers and get a higher paying job, then it was that I wasn't skilled enough to leave. It was a bit baffling as I left with 4 publications after 4 years (3 first/cofirst author), and 3 out of 4 in high impact factor journals. I think it was just seeing that level of greed to squeeze out as much work out of me while not caring about my future prospects.
I am assuming I do as my post doc also watched me the whole time, but I am definitely going to be more cautious with re-suspending my cells as someone else has also mentioned that! I currently re-suspend the cells with a 1000ul pipette and I usually have about 1-2ml of cells. Would you recommend anything in regards to how I re-suspend the cells?
I think also after you put them in the plate, do you swirl them well. One thing is if a you have a high concentration of cells in the center of the well this can also cause issues as they come together and adhere. I'm just speaking from my experience with iPSCs. Even if you mix before, just handling the plate might make the cells go to the center.
Oh you’re right I put the cells in drops spread around the well and I swirl them after but I do not swirl them directly after I’ve put them in the incubator and that might’ve been the problem! 🥲
Just be aware of that. Give the plate some figure 8s after carrying them to the incubator. Even the movement of carrying them might make them move to the center. I hope it is just a simple fix like this, but I would check the other things as well to be sure.
Oh, and by the way, you do need the raw disks to store the data, and you need a system to manage those disks (nas, storage server, etc),
Thanks for this. It gives me a lot to think about and look into. Redundancy is what we are still discussing how to be done (a division-wide system to interface with as an option). I think the small files is definitely an issue that we were trying to consider the best way to deal with.
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Thanks, I will be looking at their offerings and get some quotes.
One of my favourites. Something about that bottle just does wonders for me and I haven't found another Glenfiddich that does the same
I agree, it's also one of my favorites, something about it. Just the flavors and everything is good and balanced for me.
I have 6 unopened. 3 are the same bottle that I go through a lot, and 3 are new ones that I want to try. But I have 71 open bottles, half of which are low and will need replacing soon if I can afford lol.
I just bought it the other day from Costco for $81. I can hardly ever find it at any of my local shops.
I am! Living in the US, I try to bring some Limeburners or Hellyers Rd back with me after each trip.
I was excited for the Hellyers Pinot Noir but was not a big fan. I haven't tried their other bottles, which other ones do you recommend?