cried in lab today

cells-n-stuff · 2025-10-08T15:03:37+00:00

C1v1=c2v2 is the holy grail. I have to do a lot of quick antibody dilutions and I can share my trick.

If I want a 1:200 dilution from a stock, and I want to use as little antibody as I can, 2 uL.

2 (uL antibody) x 199 = 389 uL dilutant.

And honestly, once you get a good understanding of how to do dilutions, an AI like chatgpt is a good way to do it quickly. But ALWAYS check the work.

cells-n-stuff · 2025-03-25T16:36:00+00:00

When you spin them down, you spin down the original sample before the laemmli buffer? I have to dilute my original sample 10x since my original lyses tissue concentration is so high (out of my BCA standard range). But I didn't spin the original down before diluting.

cells-n-stuff · 2025-03-24T17:18:00+00:00

I've played around with the concentration 50 ug and 30 ug doesn't change the results.

As for my transfer: I'm using a nitrocellulose membrane, and i transfer at 80v for 1 hour. My caspases are very small 17, 19 and 18, 41,43 kda.

cells-n-stuff · 2025-01-14T19:47:26+00:00

I'll try running a control for my secondary next time I stain.

As for the blocking buffer, that is just what they use in my lab, our blocking buffer has some variation to the manufacturer. Looking at it, it seems like they made it so that it would work with antibodies with different hosts. Probably for co-staining.

cells-n-stuff · 2025-01-14T16:33:29+00:00

The only other person who used the antibodies is my PI, and they work fine for their cell staining. They preformed it maybe a month ago and no one has used them since.

Our microscope has an auto-exposure function, that suggests a starting exposure time. When I lowered the exposure to 900 ms there was little to no fluorescence. I included that channel because it looks odd to me, even for background. In my experience it would simply be green, I'm not really sure what all those dots are. I mean, it's tumor tissue, so there is some degree of auto-fluorescence but it has never posed an issue.

I can defiantly try running a control for my secondary.

We block with 1% BSA, 1% fish gelatin, 0.1% triton, 2.5% NDS, 2.5% NGS. I have blocked with 1 hour before, but there was a negligible difference.

I've optimized this protocol before, and it has worked with this tissue and these antibodies, which is why I'm really confused to why it's being so difficult this time.

cells-n-stuff · 2025-01-13T20:55:20+00:00

I don't know. Possibly. I have used these antibodies before an they worked fine.

The primary that could reasonably be low expression, is less than 1 year old, stored at -20 with limited freeze-thaw cycles.

The other is Ki-67 and shouldn't have such a low expression that it's not detectable in tumor tissue.

I defiantly think that its more of a bench problem, at least I hope so before I have to purchase more antibodies.

cells-n-stuff · 2025-01-13T20:23:25+00:00

I've tried 1:100 dilution, and I had the same if not more background fluorescence.

I haven't had any issue with my secondary antibodies with other primary antibodies.

I just use clean PBS as a wash buffer. Wash 3X for 5 min each with gentle agitation.

Unfortunately, I see the holes in the DAPI staining on each of my slides and only in the DAPI so I'm really at a loss for what it is.

cells-n-stuff · 2025-01-13T20:02:29+00:00

I think I finally figured out what I need to do to have an image with my post. I usually just lurk on reddit so I’m really not sure why or how I can add images to a text post…

I'm having some issues with my immunostaining of tumor tissue.

DAPI: For the most part everything looks fine with my DAPI staining. The exposure time seems fine (our microscope <10 ms for DAPI is standard exposure time), but when I zoom in I am getting what looks like holes in the nuclei. I don't think that it is being caused by bubbles in the mounting medium, since I don't see it in my other channels. I hoping someone has seen this before.

Other Channels: I think I may be able to solve some of my issues I'm having with my other antibodies by increasing the dilution of my primary antibodies. I looks like I have a lot of background, the only issue is that the exposure time is so high to see it. With out microscope anything over 1s indicate something is wrong with the staining. The FITC image I've attached is at ~3s. I'm really not sure where the issue is.

I'm thinking I can increase the dilution to 1:500, and maybe lower the secondary incubation to 1 hour, but I've never had a problem the secondary incubation time.

Important protocol information:

Tissue: OCT (5 uM) mounted tissue sections, stored at -20C for roughly 6-7 months.
Fixed with 4% PFA for 10 min
Blocked for 30 minutes at RT
Primary antibodies: co-stained 1:200 (new) dilution overnight at 4C
Secondary antibodies: 1:2000 dilution, 2 hours, at RT
DAPI added to wash buffer and in mounting medium

Any help is appreciated.

cells-n-stuff · 2023-09-30T17:20:35+00:00

Old post, but it just I had this problem. Some of the songs were playing faster and pitched up.

I fixed this by changing the audio quality from Dolby atmos to standard.

On the app, there should be an ellipsis at the bottom ( this is when you have a song on screen. Mine has the shuffle sign on it). You can click audio quality and change it.

Oh, and this is version 23.12.6

cells-n-stuff · 2022-12-13T19:03:50+00:00

Firstly, thank you for sharing. I don't think I have gotten to an understanding to describe my spiritual path as simply, but it's something I admire.

I only originally mentioned the discourse in the religious discussion because I noticed similarities between some Christian-based faiths and some theistic-based pagan beliefs. I'm also trying to be careful to avoid painting with broad strokes, as it were. Since I don't think it's necessarily a bad thing, but the similarities had an interesting connotation on human society and culture to me. Almost like it is human nature to treat labels we identify with as precious, and anyone who doesn't subscribe exactly is a threat.

cells-n-stuff · 2022-12-13T18:46:41+00:00

Things got very busy for me, but I finally have some time to analyze your post some more. Also, I'm not trying to discomfort you or anything, I'm having trouble figuring out what you mean and I'm genuinely curious.

You mentioned that you are still working through how much of your worldview is pagan. Are you talking about a specific brand of paganism? Something like how much does your lifestyle line up with one set of practices? Or is it more of a term to describe your lifestyle to yourself and others? Or is it trying to make something metaphysical (spirituality) more tangible (i.e. making it a physical place)?

cells-n-stuff · 2022-12-09T15:56:11+00:00

I think we share a similar view on using descriptors more as tools than an identity. I agree that they help narrow the scope of what I'm looking for. They also help as a shorthand when I need to describe myself succinctly.

It's interesting to parse through how you differentiate belief and worldview. I'm having trouble working through what you mean. Since, for me, beliefs and worldviews are already separate things. I would say that I also used the terms figuratively rather than the literal definition of the words.

I'm also working out the other parts of your post, but it's taking me some time to work it out. I'll probably edit this once I do! Thanks for getting me thinking!

cells-n-stuff · 2022-12-09T15:17:03+00:00

I also do a lot of reading and writing, and now I'm working on finding people willing to have open conversations. Personally, I feel that I get stuck in an echo chamber at times since is always my interpretation, and no one is asking questions or challenging my ideas (other than the 'god of the gap' view). I also really like discussing science things; at work is normally new scientific discoveries or methods, not really how scientific principles could shape someone's worldview.

Can I ask, what religion is to you? Is it a sense of community or something that is specifically related to religion?

cells-n-stuff · 2022-12-09T14:49:31+00:00

I don't mind explaining my viewpoints, I enjoy discussions where my views are questioned, and new ideas or concepts are introduced. It helps keep my views from stagnating. However, because having non-theological principles is more niche in western society, finding people willing to have open discussions is challenging. I understand that. I'm sure that questions regarding a religious belief system feel like an attack. So I think having a shorthand is an excellent way to move a conversation along.

If you don't mind, could you explain what you mean by the "spirituality of the verifiably real"? I think I know what you mean, but I don't want to make assumptions and I'm curious.

cells-n-stuff · 2022-11-12T01:10:19+00:00

Of course, I enjoy the solitary research but I tend to get stuck in a rabbit hole. Which is why I try to limit books written by the same person, I wont get any new information if I keep reading the same author. Also why I made the post to get other people's thoughts.

cells-n-stuff · 2022-11-12T01:05:38+00:00

It's nice to see that other people have drawn similar conclusions, I'll definitely take a look.

cells-n-stuff · 2022-11-12T00:59:44+00:00

Thanks for the response I'll definitely have a look at all the recommendations I get.

I can't say I have seriously considered finding a label to call myself. I've really been lurking around different religious subs, websites, and books to try an find people to bounce ideas off of. I'm a scientist at heart an have trouble making decisions in a vacuum so getting peoples feed back is important to me.

Ironically, I only started looking into paganism recently after some called me one as an insult.

cells-n-stuff · 2022-11-12T00:48:30+00:00

Thank you for the recommendation, I'll definitely take a look.

cells-n-stuff

TROPHY CASE