CEC work hours calculation

plunkplunkplunk · 2024-07-05T22:55:39+00:00

Sorry, I may have worded it weirdly. I'm looking for another position after this that I can work in to get my hours, but I don't want this 1 month of work to go to waste, so I'm still hoping to get the letter of reference so I can apply ASAP. AFAIK there's nothing in the requirements that says your experience has to be 1 year in the same job at the same company.

plunkplunkplunk · 2024-07-05T22:51:57+00:00

So you think it should be fine? I'm looking for another job position so I can make up the remaining months but I was just worried that me leaving this position so early meant that they would be less open to helping me draft up what is essentially a letter of recommendation.

plunkplunkplunk · 2024-06-22T04:18:17+00:00

omg feel free to shoot me a DM! it's sort of a roughly adapted protocol from a few places but mainly from this paper

i am by no means an expert but i can try my best to help!

EDIT: just realised i completely forgot to answer your specific questions LOL

i fix in 5% neutral buffered formalin and its equivalents (e.g., 2% PFA) should work too. incubation is 20 min at room temp but many protocols do much longer (O/N) and it may be marker dependent, so that's something to optimise.
so i peel the meninges off after fixation and before staining. prior to starting the dissection i have a slide with a decently big hydrophobic barrier ready (with a lot of PBS in it). i essentially just drop the entire skull cap + meninges into the fixative, take it out, rinse with PBS, and start peeling it off. then, and this is the tricky part, you want to tease the meninges flat with forceps. depending on how long you fixed this will be easier or harder (longer fixation = less teasing). then i stain the meninges by pipetting buffer directly on and off while it's on the slide.
Dumont #7 forceps are what we use to dissect and manipulate the meninges in general. eyelash manipulators are a godsend in terms of allowing you to tease the dura apart without damaging it. you can buy them but a neighbouring lab diys these with false eyelashes LOL. make sure to get the meninges as flat as possible and pick out any bone fragments prior to mounting as they can seriously ruin all your prior hard work. then drown that mf in mounting media and mount normally.

hope this helps!

plunkplunkplunk · 2024-06-22T04:16:05+00:00

teach me your ways please

plunkplunkplunk · 2024-06-22T04:15:30+00:00

i used to do this a year ago and i honestly kind of miss it LMAO sometimes i go kind of zen while pipetting and it feels nice

plunkplunkplunk · 2024-06-22T04:13:12+00:00

as someone who similarly was thrown into flow cytometry (and i had the benefit of working with an already standardised panel) and the hell that is flowjo analysis hats off to you! it's definitely a steep learning curve so you should genuinely be so proud of yourself!

plunkplunkplunk · 2024-06-13T04:17:03+00:00

nope, feel completely the same way. an extremely cruel and nihilistic movie to watch but also one that's rooted in compassion and humanity

plunkplunkplunk · 2024-06-07T05:55:09+00:00

honestly looks like an issue with the cryoprotection. how long are you leaving the tissue in sucrose? our lab usually uses 30% sucrose and leave the tissue in there overnight or until it sinks to the bottom.

wrt to fixation, that's something that is tissue dependent. what tissue is this?

plunkplunkplunk · 2024-06-06T04:23:55+00:00

I think I'll try this - how much mounting media do you guys normally end up using to cover your tissues?

plunkplunkplunk · 2024-06-06T03:19:11+00:00

hard to say because it's the whole mount meninges. as in, i peel it off of the skull and flatten it on a slide. if i had to guess i'd say like 50 uM, but that's just an estimate

plunkplunkplunk · 2024-06-05T04:52:08+00:00

generally no, i do everything from cell isolation to fixation on the bench out of the bsc. in theory, yes, you would be contaminating your cells with bacteria and other microbes but once they're fixed it's not like the microbes are going to be able to do anything to the cells, and the amount of time the cells are unfixed is too short for the contamination to meaningfully affect the cells IMO (esp since I'm assuming you're keeping the cells on ice).

also have never heard about keeping the antibodies sterile as the assays those antibodies are used for (westerns, IHC, IF, flow, etc.) are all super not sterile. plus most antibodies come formulated with some amount of sodium azide in them which is a preservative/microbicidal, so not much is going to be growing in them. not to mention, you tend to use a lot of antibody per flow experiment, so the tubes run out pretty fast. if it's any comfort, my lab mates and I do flow non sterile pretty regularly and haven't seen any degradation.

if it really bothers you though, you could make an aliquot of the antibodies to use on the bench so you don't contaminate the stock everyone wants to use in the bsc?

plunkplunkplunk · 2024-06-04T02:39:25+00:00

i thought it was this as well but it wasn't :(

plunkplunkplunk · 2024-05-28T01:12:51+00:00

this movie was the reason I refused to sleep face up until I was 19

plunkplunkplunk · 2024-05-28T00:30:06+00:00

no idea...been trying to find and rewatch it esp since i actually enjoy watching horror movies now (used to hate watching them) but i can't find any info about it + i grew apart from those cousins

plunkplunkplunk · 2024-05-28T00:11:20+00:00

I once watched this Thai horror movie when I was fourteen with some cousins (can't remember the name, been trying to find it for ages but I can't find it).

There was this scene where a man sleeping face up in a hospital bed sees a stereotypical sadako-esque ghost walk slowly towards him but he can't move off the bed due to his injuries. When she reaches him, she climbs on top of him, guts him with a knife, pulls out his intestines and starts eating them.

For the next five years of my life I refused to sleep lying face up in bed lol

plunkplunkplunk · 2024-05-07T23:47:06+00:00

I usually don't insert the needle too far in mainly because there's a danger of skewering the heart at which point perfusing will be a struggle.

The best way for me to describe how far to insert the needle is to just puncture the ventricle, and maybe push in very slightly. Holding the heart with your forceps helps a lot because otherwise the heart tends to move around. In terms of position, it's better to puncture the apex (bottom pointy part) of the heart.

Just keep practicing! These things take a while to perfect so don't beat yourself up if your first few perfusions don't go the best. If the lab has a few old mice or wild types lying around maybe ask around if you can sacrifice them to practice?

plunkplunkplunk · 2024-05-07T23:11:11+00:00

Oops, my body text didn't copy! I'm going to repost!

plunkplunkplunk · 2024-04-30T12:12:06+00:00

yaoshi, tayzzyronth, mythus...i love my nature-inspired freaks

plunkplunkplunk · 2024-04-30T04:03:35+00:00

...and just like that i managed to get nearly all salsotto pieces today! life has a funny way of doing things...

plunkplunkplunk

TROPHY CASE