MErc44

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Immuno staining is f$cking weak. I'm in the lab after hours and all the postdocs are at home and here I am adding a block before I can add primary antibody. I can't wait until I graduate so i don't have to do anymore of this lab work. At least I can waste my time on the computer
 

TTSD

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Originally posted by MErc44
Immuno staining is f$cking weak. I'm in the lab after hours and all the postdocs are at home and here I am adding a block before I can add primary antibody. I can't wait until I graduate so i don't have to do anymore of this lab work. At least I can waste my time on the computer

A-freakin-men! Having to sit there 9-18 hours at a time.. NOT COOL MAN!! NOT COOL!! Especially when it doesn't work.
 

Treg

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Why not just block overnight at 4 C? I like western blots because you can time any of the steps-run get overnight, or transfer overnight, or block overnight, etc. It is just the washing that is a bit tedious.

Treg
:)
 
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SticknRudder

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If I can avoid westerns by doing northerns, I will any day of the week. Blech-- sh!t ass!
 

jamnx

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its all about multitasking. go inject some mice or order pizza while you block.
 

MErc44

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yea, i finished the block then added primary antibody which i put at 4 degrees overnight. I am actually doing cancer research so I only work with cancer cell lines, I don't harvest the antibodies myself. Even worse than immuno staining is diluting the compounds we test. While the people I work with are talented the lab is so disorganized that I spend at least a half an hour before an experiment looking for supplies and reagents.
 

mdforme

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damn my PI always gives me PLENTY of things to do during incubations. It is sooo weak. Can't even get my SDN fix....
 

mdforme

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I have in other labs but here I'm a pair of hands....
boring
 

MErc44

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it sounded cool but then I realized how science was done and that I wanted no part of it. Since that realization which happened sometime last year I have only stayed because I don't quit what I start and I get a 2 or 3 unit A each quarter which has boosted my gpa. I used research as a tool to get into med school, I got in, the tool no longer serves a purpose but I remain. F%ck.
 

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Originally posted by MErc44
it sounded cool but then I realized how science was done and that I wanted no part of it.

Amen! I can't wait for medical school because I realized how much I hate being a lab researcher. I just can't handle a job where you fail 90% of the time no matter how good you are. Finally a thread to vent about stupid stupid research!
:smuggrin:
 
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Originally posted by Treg
Why not just block overnight at 4 C?

I know some of my antibodies' spec sheets said to use primary overnight at 4C. This also helped to preserve the antibody longer, versus doing it at room temperature (or so I'm told). So us lab techs always had to block them before we could leave or we'd blow a day.

Ahhhh, am I looking forward to my PhD? At this point, not really :) Not like I like med school though. The third and fourth year MD/PhDs tell me grad school is like a vacation compared to what we're going through now and especially clinics in second year.
 

Celestron2000

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Working as a lab tech pretty much convinced me that research was what I did NOT want to do. :scared:
Decided to apply to medschool (there's more to it than that, but well, this was my motivation.)
 

Chrisobean

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i dont understand why your immuno takes so long to do, just to get to the primary.
if im doing animal chunks, i dissect in the morning, fix and wash and block for at most 4h and put the primary right before i leave, and i leave at 430...
for cells, i block for an hour tops, and even that is a long time, sometimes all you need is 20min. i can do a whole staining in one day if i start in the morning, otherwise i take my time and leave the primary ON also.

what am i missing?
 

MErc44

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you see, I am a lazy senior who is taking a full load of classes so at most I have two or three hours a day, it takes about two hours or an hour and a half before I can add primary so I generally have to start it late in the day after my classes. If I didn't have school it would be no problem
 

Chrisobean

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thanks for the clarification.

by reading your posts, i thought i was doing this super-secret super-fast immuno while you guys were slaving over it for days... hehe. guess i am not that cool :thumbup:
 

MErc44

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F%ck am I supposed to wash with water after the block, before I add primary, and wash again before I add secondary?
 

Chrisobean

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i dont wash after blocking, i just remove it and add the primary (which is diluted in fresh blocking solution)
always wash after primary (reduces background)
debatable whether it is necessary to block AGAIN after primary... i do it with tissue but not with cells. secondary is again diluted in blocking solution, then wash again. this wash is more important, it GREATLY reduces background. mount, and yippe your done!

speaking of, i have to go wash out my secondary right now :)
 

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I found out that research wasn't for me after about 6 months. The prof who "runs" the lab (I say "runs" because it APPREARS to the average person that he knows what's going on) had 200 different isolated compounds to screen on breast and colon cancer lines. Anyone who has ever done screening, knows what a pain in the arse this is. So time consuming to run an MTS assay and THEN a vital cell count after 5 days.

anyway, my prof is a brilliant organic chemist but knows absolutely NOTHING about cell biology. He doesn't get the fact that 90% of compounds are going to KILL human cells at concentrations above 10^-4 M concentrations. He doesn't understand the concept of dosage and toxicity to normal cell metabolism. He'll get all excited when I find a compound that works at insanely-high concentrations but fails to do anything in the micromolar range. I'll have to explain to him why it's not a good compound and he'll just give me a blank stare.....

I got really tired very quickly of being under the guidance of someone so inept at biology and yet such a talented chemist. It ruined my taste for any more research.
 

celticmists18

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hello my fellow lab rats!
I'm currently bored out of my mind waiting for my cells to dettach . . . I HATE, HATE, HATE research, so much so that I am actually looking forward to starting med school. Never thought I would see the day when I was counting the days till school started not ended! Is anyone else doing research full-time?
 

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Originally posted by celticmists18
hello my fellow lab rats!
I'm currently bored out of my mind waiting for my cells to dettach . . . I HATE, HATE, HATE research, so much so that I am actually looking forward to starting med school. Never thought I would see the day when I was counting the days till school started not ended! Is anyone else doing research full-time?

Yeah me too! I graduated early and started working for my professor full-time this year. Boy will I be glad to start medical school and stop this torture. The main thing that I hate so much is that my experiments fail most of the time even though everything appeared to be going really well. And the flexibility of being a lab tech is cool, but I would rather come in to work and have things that need to get done during my hours and then go home when the day is over. Here I have to decide for myself what to do next and then regroup after it all goes to crap. It feels twice as bad because all of the other people seem to love it or something and they work 10 or more hours a day! Gah! Anyway, I guess I am done venting. I am glad there are more of us out there ;)
 

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Boy...I sure can't wait until I start my first semester as an undergraduate researcher in the fall! lol...Reading this thread is actually pretty depressing...
 

Celestron2000

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Hey celticmists18 looks like we have a lot in common (I'm a big Tolkien fan as well.) Sure you're not comming to Jeff 2nd look day?
I've been working full time as a labtech for the past 3 years!
It does have it's advantages, but I can't wait to move on.
 

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Undergrads are the lowest common denominators of research. It can be pretty discouraging at times.
 

MErc44

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you know what else blows? Looking for ****, and working in a lab when noone tells you how to perform the procedure. I get a protocol with fragmented sentences and am told to carry out an experiment. I swear if you have to do bench work as a med student to get an orthopedic surgery residency I will seriously reconsider... EM sounds pretty good right now
 

premed

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There are alot of us full-time lab rats between undergrad and med school. Unfortunately I don't know when med school will begin. :( Anyways, I've been doing IHC for 4 years and it is not very time consuming. First day, I spend a little over 1 hour in which I am actually working about 10 min. The other 50 minutes I'm on SDN. The second day takes about 2 hours. There are ALOT of unecessary steps people tend to do for IHC. Someone posted blocking twice?!? One 30 minute blocking is more than enough. Besides, the primary and secondary should both be diluted in blocking solution. Anyways, if anyone needs help in IHC feel free to PM...research is 1 thing that I know. :)
 

Thundrstorm

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Originally posted by BerkeleyPremed
Boy...I sure can't wait until I start my first semester as an undergraduate researcher in the fall! lol...Reading this thread is actually pretty depressing...

If it helps, I've been doing reasearch for a year and I still love it, even the repetitive things (well, except maybe cell counts). I'm only working part time though, and I mostly work with cell culture, so I guess I don't have the complaints of some of the other posters. We'll see if I still love it when my hours increase next year as I do an independent project and write a thesis. :)

I'll be taking a year off before med school to do research full time, so I hope I maintain my geeky enthusiasm.
 

hamhamfan

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Originally posted by premed
There are alot of us full-time lab rats between undergrad and med school. Unfortunately I don't know when med school will begin. :( Anyways, I've been doing IHC for 4 years and it is not very time consuming. First day, I spend a little over 1 hour in which I am actually working about 10 min. The other 50 minutes I'm on SDN. The second day takes about 2 hours. There are ALOT of unecessary steps people tend to do for IHC. Someone posted blocking twice?!? One 30 minute blocking is more than enough. Besides, the primary and secondary should both be diluted in blocking solution. Anyways, if anyone needs help in IHC feel free to PM...research is 1 thing that I know. :)

Hi, could you help me with this vector red stuff? Not even the lab techs have much experience with it and so I've been kind of stabbing at it.

Problem 1: background. I used blocker for 30 minutes, but there still seems to be some background.

Problem 2: the last time I used vector red, the whole thing didn't even work out (like 6 hours down the drain). Do you know what the left-over solution is supposed to look like after 20 minutes? The solution didn't seem as dark as usual, or maybe I'm thinking too hard.

It didn't work before, but that was because I didn't adjust the pH of the Tris to 8.2-8.5.


Thanks!
 

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Just in case anyone else cares to know...

Hey Ham Fan,

First of all, can you answer some questions for me:

1. What tissue or cells are you staining (I'm assuming tissue staining via ABC (Avidin-Biotin Complex) substrate since you are using Vector Red)?

2. Frozen or paraffin sections?

3. What are you using for blocker?

Problem 1: What do you mean by background? When adding your Vector Red (BTW I've used Vector NovaRED...I think you are referring to the same substrate) I usually let it stain for 20 minutes. The binding affinity of these color stains are not as good as DAB. Regardless, there will always be some background staining. I think that the problem may be that you are using too high of a concentration of primary antibody. By oversaturating the primary antibody, you are getting non-specific binding and ultimately getting staining on other areas which appears to be background staining. I suggest you do 2 things. First you should get 1 slide and skip blocking, primary, secondary, and ABC. Just fix, perform antigen retrieval (if you are using paraffin sections), wash through PBS or TBS and add the Vector Red. View under a microscope (by eye, you will almost always see a red hue to the tumor...this means nothing) if you see positive staining, you have endogenous peroxidases and will need to block them. The best way to do this is to add 0.3% H2O2 before your blocking step. If this is the case let me know...I will give you more advice. The second thing I suggest is that you perform a titration analysis, which is a must for good IHC staining when using a new antibody. If you are using anti-serum, I would suggest 1:10, 1:50, 1:100, 1:200 dilutions of the primary antibody. Run them all together making sure you add a different dilution of the antisera to each of your slides and make sure they don't mix. If using anti-IgG, try dilutions of .5 ug/mL, 1 ug/mL, 5 ug/mL, and 10 ug/mL. I usually have best success with 1-2 ug/mL. If you are using a self-generated antibody, let me know...I have more advice for this.

Problem 2: Again, I've only used Vector NovaRED before, but I think we're referring to the same product. From what I remember, after making the solution, it is clear. However when you add it to peroxidases it will turn red. The left over solution is still clear.

I have no idea what you are using the Tris for? Are you making some of the solutions on your own? I buy most of my substrates from Vector Labs and make my own blocking solutions and generate my own polyclonal antibodies by immunizing rabbits...fun stuff.

Sorry this was so long, feel free to PM me. It took me 4 years to perfect IHC staining and I had to learn alot of it on my own which really sucks. :mad: Good luck!
 
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