Deadline Check-in

So here it is Monday. I am supposed to be *done* with a draft of my ManuBeast.

I am happy to say that I made a HUUUUUUUUUUGGGEEE dent in it over the last week.

I am not so happy to say that the panels for a figure that I thought I had in the bag were not forthcoming during that time due to a sample inventory snafu. I used up my last remaining samples that would have served that purpose during the Great Trouble-shooting Epic, and did not generate more during that time. Ooops. So I've gotta do that now, which isn't too much trouble, I just would prefer to cross that off the list.

In other news, I was supposed to spend yesterday polishing up the last bits of text (since I got the bugger all written last week - woohoo!), and today inserting references that I haven't already. Unfortunately those efforts have been dampened by skull-splitting sinus pressure and a low-grade fever. I tried. I really did. But after spending several hours (and several attempts) trying to merely comprehend the genius that I had put on the page last week, and having exactly zero success, I've conceded. I instead spent today (and much of yesterday) horizontal (because vertical makes my head explode), accumulating a K2 of tissue around the couch, drinking tea and broth, and watching crappy films like " Lara Croft: Tomb Raider". (I actually really like this movie. It's not great cinema, but I kind of wish I could be her, and the mindless escapism is exactly what a head cold calls for. Sue me.)

So while I am not really truly done with the fucking thing yet, I made some progress that I can be proud of, and the end is so close I can taste it. Or I could do, if my sense of smell were not completely obliterated. I'll probably be quiet for a little while longer until it's really finished.

Please excuse my absence....

Folks, you will not be hearing much from me in the near future. I took my weekend - it was good. I am feeling mostly recharged and I am motivated to get a finished draft of the ManuBeast to GrAdvisor by the end of the month. That's a week from today (shit!). So I am directing all of my writing efforts between then and now toward that end.

See you on the flip side.

Reality Check

I am not taking very good care of myself. I know better. It has been over two weeks since I last had a day off. I have been doing the very same thing every day in the lab since then. It is working; I'm not repeating things because the experiment is failing. It's just a very very time-consuming protocol encompassing lots and lots of samples. I managed to compile all of these data into a MegaFigure yesterday. I presented it to GrAdvisor in our pow-wow about the ManuBeast, and as I expected, it softened him to hear my concerns about the BullshitExperiment (which is a waste of my time and should not go into the ManuBeast). Well, at least not on the first submission. He wants to change tacks on the BullshitExperiment, which I will continue working on while the ManuBeast is out for review. If we get anything out of the BullshitExperiment (unlikely in my opinion) we can include it in the resubmission. He is still not hearing my concerns about the fact that it will be difficult to conclude anything from the expected negative result, but I'm happy to take this one off the table until we submit at least. This is not a victory, but an acceptable compromise. It thought that the MegaFigure was going to be a bit of a tough sell. It is not fully annotated yet (which makes it hard to impress GrAdvisor), but it is giving me a very interesting big picture of my Loogly-fluglies. Things we didn't know before. Things that are changing our model. I like this. He seems to like this. I like the momentum I've had going here for a while. I feel like I've gotten more done in the last month or so than I have in the last 3 months combined.

But I am exhausted. I have been for some time. I haven't slept well since July. I wake up at strange hours and when I do sleep I have nightmares. I am emotional eater (not a good thing when you're stressed) and I feel like I have neither the time nor the energy to get the exercise I should to counteract both the over-eating and the anxiety. My nerves are raw, my hands shake, I feel like I *need* a drink just to settle myself at the end of the day. There is too much hair stuck in my hairbrush when I comb it out in the morning. My shoulders are so tight that it's difficult to pull my hoodie off over my head. I have arguments with BH over stupid things not because we disagree or because I am being irritable, but because I am utterly failing to communicate well - I can't get my brain to process the things that I mean into words that express them. This morning I had a little meltdown. I laid down and cried because I was just....so....tired....and I still have more work to do.

This is not good - not in the long or the short term. Yesterday I stayed home, did a little work on the MegaFigure, and some laundry, and basically took a mental health day. That helped some but it hasn't fixed it. I need to walk away from the lab and the manuscript for a few days and take care of my body and my brain instead of sacrificing these things for the Almighty Publication.

I can't do it just yet. We've got some major analyses planned for tomorrow that's been in the works for three weeks now. It will run for several days. So I'm planning on getting that going and then coming in to just make sure that the automation is running on Saturday.

Other than that, no science this weekend. That's my promise to myself. Instead I would like to go to the Farmer's Market. I would like to go to the pub and watch some rugby and drink some beer with friends. I would like to go see the not-so-new-anymore Harry Potter film. I would like to go to the museum before this exhibit I've been wanting to see moves on. I would like to take DangerDog out for a much-needed run. I would like to sleep soundly, at whatever time of day pleases me. I may not do all of that but I am clearing my schedule this weekend so I can do any of those things. We'll see how that goes. After checking automation on Saturday morning I will not think about science again until Monday.

Raise the Drawbridge!!

Or, How To Conduct A Medieval Siege, 21st Century Style

Figure 1: Enemy occupation of the AA residence, taken in the moat-building stage.

1. Ingratiate oneself to the residents of the citadel that one wishes to eventually occupy. This will allow you easy access to their defenses so that you might better use them to your own advantage. In this era this is best accomplished by feigning improvements to the residents' current living situation. We recommend infrastructure improvements such as road resurfacing or water main repair (we'll come to this later).
2. Foster a sense of trust in the serfdom by providing formal and official looking documents asserting your good intentions and promising minimal inconvenience. It is critically important to buy yourself as much goodwill as possible at this point, as this will prolong the period during which the occupants of your target remain confused about your actual intentions. Stating an end date for the project on formal letterhead usually does the trick.
3. Let the games begin! Remember, a siege is a test of patience and a battle of wills. It is important to hit your target not just physically, but emotionally as well. Take every opportunity, no matter how small, to irritate or confuse the inhabitants - the sooner they crack, the sooner you win! Good first round strategies include re-routing street traffic frequently and irregularly so that it is difficult for the inhabitants to come and go from their citadel. A house on a street corner is best for optimal effect. Keeping them guessing also subtly reinforces a sense of futility in their predicament.
   
4. Give the illusion that you're still on their side. Again, this is psychological warfare; sowing confusion is key. Damage their property then claim ignorance, then innocence, then apologize and make noises that you might replace or even improve it...when you're "done". Ha!
5. Time to up the ante - start cutting off resources. Shutting off water service or electricity without warning is always a winner. But don't get greedy just yet. Make sure that these disruptions are merely annoyances at first - the goal is to wear down and distract your target without triggering any defensive maneuvers while you position yourself - don't deny services for too long or they will start to suspect.
6. Once you have utilized the above tactics to move your troops and resources into attack position with minimal resistance from the natives, you may commence a full-scale siege.
7. Build a moat! The deeper the better. Opening a nearby fire hydrant will increase impassibility. Again, corner houses are best, bonus points if other access points are geographically restricted - close neighbors and no alleyways make your isolation tactics even easier to employ. This is where it comes in handy to have operated under the guise of subterranean infrastructure improvement. They didn't suspect a thing when you moved in all that digging machinery, did they?
8. Increase the psychological assault. Sleep deprivation techniques are highly effective. Some have employed loud noise such as blaring rock music with great success. We recommend jackhammers and shaking the foundations of the citadel at obscene hours of the morning, or both at once. Even better if you can manage to have the water cut off during the residents' early morning rituals like showering or making coffee - this will cause no small amount of consternation and despair (you'll know you achieved the desired result when shrieks coming from the second story bathroom can be heard over the jackhammers).
9. Further restrict transportation to and from the citadel...and if you can, add insult to injury. Build a "bridge" across your moat so that they will sink their cars into the mire when they try to cross it. If they still manage to get in and out, make it such that they can only park on one specific section of the street, then re-route traffic and have their cars ticketed and towed. Now that you have completely cut off access to the citadel it is impossible for the residents to bring in resources or reinforcements - from this point forward it's just a waiting game.
   
10. Be patient. Stay strong. As you approach, then surpass your aforementioned "end date" with no actual end in sight, residents will start to lose hope. By continuing to employ these tactics over a prolonged period of time you will completely wear down the natives' will to resist. When they finally surrender victory is yours!

On Dealing with Parents (and other people who don't "get it")

DGT has a post up on relating to non-sciencey parents. I think that her oregano analogy probably works well for those non-sciencey folks who are trying to get a handle on what you do and why you're good at it (or not, I guess, but do parents really even consider this possibility?).

I've had some recent frustrations with my own non-sciencey set. They are well-meaning folks but in spite of my best efforts to explain to them what is going on in my life, in my research, in my career, they're just not getting it.

I'm fairly fortunate to have parents that do "get it" with respect to the value of education (they're probably where I got my ideas about this after all). Not everyone is so lucky in this respect. They aren't asking me what I'm going to still more school for, nor suggesting that all this education is just going to give me "big ideas".

Both my parents have bachelors' degrees in a rather obscure applied engineering sub-discipline. They both started working in that obscure sub-discipline right out of college. My dad has continued to work in the same industry ever since, though not always in an engineering capacity. My mom quit to have and raise kids, then went back to school, earned a Masters' in Education, and now teaches middle school science. So they're not terribly far removed from higher education and sciencey/engineering type fields. But neither of them have any direct experience with the kind of science I do, or the kind of degree I'm getting. They have never worked in a capacity where they need to publish peer-reviewed papers for career survival, nor have their educational experiences included an open-ended time frame like my Ph.D. in Mystery Bioscience.

I get the usual interrogation about when I will actually be finished. It doesn't matter how many times I have explained that my Ph.D. is dependent upon my success at publishing relevant papers, which is in turn dependent upon the success of my experiments, which is in turn dependent upon how well I design and execute them in addition to myriad other factors outside of my control like whether or not our lasers have burnt out, the relative amorousness of my experimental subjects, the alignment of Jupiter and Neptune, and which direction the wind is blowing. My mother remains convinced that I do know when I'll be finishing but that I'm just not telling her because I don't want her to come to my defense (which isn't even true - I don't know where she got this idea). Like my dissertation and I are planning to elope or something. After several years of this infuriating questioning, I finially snapped and told her I would hang up the phone the next time she asked me the dreaded so-when-are-you-going-to-finish question. I mitigated this offense by promising that she would be the seventh to know (after myself, my advisor, and my committee members) if when I ever establish a defense date. This is a common experience for most grad students in my field. The question never really goes away, even after threats (no, I've never made good on that threat and hung up on my mother though she persists in asking The Question). The well-adapted learn to stop hearing it.

I don't fault my mother for asking this question. I know it comes from concern and a desire to be supportive. I'm very fortunate that I've always had parents who take an interest in my success. I know that's what this is about.

But sometimes I wish they could stand in my shoes, or at least really truly listen when I tell them how it is. It's not like we've never talked about what it is that I do, or how this whole Ph.D. thing really works. They are genuinely interested so they ask all the time, and frankly, we've talked it to death. But they still don't "get it". I suppose that they can't really, never having been here themselves. But it's also that they're very good at projecting onto me their own experiences. If they can find a scrap of commonality between what I'm telling them and something in their own lives then it's "Oh, your X sounds just like my Y, so This Is How It Must Be." No, mom and dad, it's not like that. You asked and I'm telling you how it is and you're just not hearing me.

My mom also falls into the trap of giving me advice on how to handle the various stresses of my career. Again, I know that she does this out of love. It pains her to know when I'm stressed out and exhausted, and she's trying to help in the best way she knows how. Which is to project the AA that she knew as a child on to AA's now adult professional person. Which is usually not very helpful.

I was speaking with her on the phone a few weeks ago when all the shit hit the fan with my experiments. You remember, the trouble-shooting epic? Yeah. I was telling her what was happening and how frustrating it feels to not be able to get anything to work. How it feels like that light at the end of the tunnel (in this case, publication of the ManuBeast) is just slipping away. Her advice: "Just do it, AA! Get it done!" My mother, the cheerleader. I know that this was the only thing that she knew to say for encouragement, but geez mom, did you even hear what I was saying about all the work I'm putting into this right now and how all of my experiments are failing? If the problem were "just doing it" I would already be done!

I was speaking with her again recently (while in the lab collecting data in the late hours of Sunday evening) and she asked how things were going with the ManuBeast. I told her that I'd been successful in my trouble shooting and things were working again, but that the next big hurdle would be to convince GrAdvisor that we should just drop BullshitExperiment for all the reasons I've previously mentioned. She said to me, "You know AA, you've always been such a perfectionist. You're always so ciritical, especially of yourself. I was just telling YourFormerElementarySchoolTeacher at the community picnic last week 'Oh, you know AA. It takes her forever to finish anything because it's never quite good enough to meet her standards. I suppose it will be the same with her Ph.D.'"

AAAAAaaaaaaaaaarrrrrggggggggggggggggghhhhhhhhhhhhhh!!!!!!!!!!!!!

First of all, I prefer "recovering perfectionist". I know that keeping impossible standards creates a lot of unnecessary stress in my life so I try to aspire to make things as good as they can possibly be, but not more so. Yes, I suppose it is true that I am a bit of a perfectionist. But you know what - that's a large part of what makes me a good scientist. Those high standards mean that I'm not going to write and submit a paper based on sub-par data. Which is a good thing, because I have to meet not only my own standards to be successful, but those of a whole boat load of other people. Journal editors, reviewers, my committee memebers, my advisor. To some extent, my success in this endeavor depends on meeting their standards. And this is a Good Thing, to have external validation of the merit of one's work. Especially when one's work provides the foundation for future understanding. Especially when one's work contrbutes to the truth, as best we can currently understand it. It's important not to do slip-shod work. This is not a college term paper, for which I can say, "making an A on this paper is not worth my sanity today. I can justify cutting myself some slack and taking a B on this one." This isn't a class where the only thing riding on my efforts is a letter on my transcript. But it's easier for her to frame it in this way. She's a teacher. This is what she knows.

I don't really know how to make it any more clear to her that my stress, the pressure I'm putting on myself, my crazy working hours....these things are not me being crazy. They are what I need to do to make this happen. It will not just take care of itself.

PSA

DrdrA has some great news! Go congratulate her.

On Courtesy

Earlier today I was working on a critical task at my computer. I could not afford to lose my place and I was focusing very intently. This sort of thing happens in laboratories. Most people have the good sense not to interrupt, or, upon realizing that they have interrupted, to wait for a better time to converse. Some people do not.

AA: [typity typity type type type click *shit!* ctrl+z typity type...]

Labmate: (shouts from end of bench bay) Hey AA!

AA: (without looking up) [type type type *fuck!* ctrl+z] Yes?

Labmate: (louder) HEY! AA!

AA: (without looking up) [type type type *goddammit!* ctrl+z typity type] Yes, Labmate??

Labmate: AA! *snaps fingers*

AA: (without looking up) [WTF!?!?!!??? type type *fuck!* ctrl+z type ] *snarl* DO NOT snap your fingers at me!

Labmate: (still not getting that the center of universe is elsewhere) Well, you have to look.

AA: (without looking up) [type type type *sonofabitch!* ctrl+z] YOU have to have a little respect for other people around here. I am in the middle of something. If you cannot be bothered to tell me what is so godalmighty important that I must stop what I am doing right this second and lose my place to see it, then it is not that important. [typity type] I am not a dog. I do not respond favorably to people snapping their fingers at me. It is incredibly disrespectful, as is your interruption. If there is something that you want me to see, it will have to wait until I am done with this, or at least at a good stopping place. [type type type *shit!* ctrl+z]

Labmate: (storms off in a huff)

I still have no idea what Labmate wanted to show me. Huh. I guess it wasn't that important.

Working for the weekend

Seriously. I really really really want to take this weekend off. There's a rugby match showing at the pub tomorrow morning (bangers and beer for breakfast - yay!), and a Shakespeare festival in the park tomorrow evening. I haven't had a good run or swim in ages, so it would be nice to do one or both of those at some point too.

But this ManuBeast figure needs to get done and I really really really want it to be finished. I am tired of thinking about it and tired of working on it and once it's submitted I have permission to write (the dissertation).

Maybe I can take tomorrow off and work on Sunday. We'll see how it goes today I guess.


***************************************************************


Non-sequiter: why is our house suddenly overrun with ants? Usually they make the mass exodus to the indoors when the ground is waterlogged, but we've been in a drought lately. We have ants trying to get into the cat food, ants in the recycling, ants in the laundry (lots of them there - WTF ants?).

I have a bit of a soft spot for ants. I used to study Atta cephalotes. They're adorable, they have weird sexual genetics, and I love the colony-as-organism social structure. These ants that are now in my home are decidedly not the peace-loving Attas, and they are taking for granted my good will towards all Formidaeans.

I have an ant bite in my armpit. It itches A LOT. I was just lying on the couch watching a movie last night and some ant was offended by my presence in my own house. So it trekked across the room, up the side of the couch, under my shirt, and it bit me. I am not amused by this.

As long as it's not a full-scale invasion I'm usually not bothered too much by them. They come in, they look around, decide there's not much to be had, and they go on their way. These ones are getting fucking territorial. I'm also a little territorial about my own house. I will tolerate the presence of arthropods if they earn their keep in mosquito control, or at the very least, mind their own business. When they start to take advantage though, I do object. Just ask the cockroaches.

Listen ants, if I step on your home (you know, the one outside in the garden), you get pissed and swarm out and bite me all over the damn place. Then I itch for weeks. Fair enough really. But you seem to have overlooked the fact that now you are in my home. I have been very tolerant of your presence thus far, but if you start getting pushy I will be forced to take action. If this escalates any further I will deploy poisons. Ants, you've been warned.

Found on Facebook

Directed evolution of a full professor

A.L. Nighter, F.N. Dorf and Steph A. Rose

Philosophical Notions About Science (USA)
Vol 2002, pp 3104-3109, October 1998

Section: Evolution

Keywords: student / postdoc / professor / academic adaptations / ethanol-induced mutations

Laboratory of Human Applied Directed Evolution, Stanley Hall, University of California, Berkeley, California 94720, USA.

Communicated by Wendell Stanley, October 10, 1998 (received for review September 26, 1998).

ABSTRACT

Success in academia is hypothesized to require specific phenotypes. In order to understand how such unusual traits arise, we used human clones to identify the molecular events that occur during the transition from graduate student to professor. A pool of graduate student clones was subjected to several rounds of random mutagenesis followed by selection on minimal money media in the absence of dental insurance. Students surviving this selection were further screened for the ability to work for long hours with vending machine snacks as a sole carbon source; clones satisfying these requirements were dubbed ‘postdocs’. In order to identify ‘assistant professors’ from amongst the postdocs, this pool was further mutagenized and screened for the ability to turn esoteric results into a 50-minute seminar. Finally, these assistant professors were evaluated for their potential to become full professors in two ways: first, they were screened for overproduction and surface display of stress proteins, such as Hsp70. Assistant professors that displayed such proteins (so-called ‘stressed-out’ mutants) were then fused to the M13 coat protein, displayed on phages and passed over a friend and family members column to identify those that were incapable of functional interactions. These were called ‘full professors’. Although these mutants arose independently, they shared striking phenotypes. These included the propensity to talk incessantly about their own research, the inability to judge accurately the time required to complete bench work, and the belief that all of their ideas constituted good thesis projects. The linkage of all of these traits suggests that these phenotypes are coordinately regulated. Preliminary experiments have identified a putative global regulator. Studies are currently being conducted to determine if overexpression of this gene product in postdocs and graduate students can speed up the graduate student to full professor evolutionary process.

*Karen Ottemann and Sharon Doyle, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, USA.

With a vengence

My mojo is back. That is all.

Motivation

...or lack thereof.

Now that I got my antibody straightened out, I need to trouble-shoot my nuclear counterstain (which worked fine during the antibody trouble-shooting but now is not working at all). I don't really know where to begin with this since there's not much to that protocol except "apply at 1:1000 in PBS during the penultimate wash", which is what I've always done and it has always worked...until now.

I can stain two slides in parallel - one from the old group and one from the new group. I expect I'll get stain on the one from the old group and not on the one from the new group, which will just be a replication of this weekend's unplanned experiment in counterstain efficacy. What will this really tell me? That I have 6 boxes of slides (new group) in which I have somehow completely stripped the sections of nucleic acids? (Is that even possible?)

I'm not really superstitious, but lately it seems like everything I touch just turns to shit. I am the anti-Midas. I can't decide if it will be more effective to stay in bed until my mojo finds its way back or soldier through and meet my bad karma quota for the century so things can get back to normal again. I feel like handing in my "credible scientist" badge. Who the hell fucks up a nuclear counterstain? Seriously, never have I sucked so badly as I have for the last two weeks.

10 Things I Like About Life

There's been a lot of negativity on this blog lately. Most of it justified. But since I've been feeling rather homicidal lately and I don't think that's very healthy, this is an exercise in positive thinking. Here is a list of 10 things I like about my life right now.

1. I have health insurance, and so does BH. If shit hits the fan with respect to our well-being I am not worried about choosing between treatment and paying the rent. I do not take this for granted.
2. The house we live in. The landlady can be a real pain in the ass at times, but we have enough space for the both of us (plus pets) to be comfortable together and to have our own space at times. We have good neighbors, and a fenced yard for DangerDog, and the landlady's permission to do whatever we like with the garden (and I have!!).
3. I have a job. It's a "job" that makes me crazy sometimes, but that's because I'm engaged in my work. Sometimes (like when I'm on the microscope at stupid o'clock) I wish I had a mindless 9-to-5, but I know that would be fatally boring for me in the long run. I like figuring stuff out, which is what I get to do every day. I like it better when it works, but you know, CareBears and tea parties and all that.
4. I don't have to drive to work. It's painfully hot here so the walk/bike ride is not always pleasant in the summer, but paying for parking and sitting in traffic would be much more painful. I like the time I have to gear up for the lab or wind down on the way home.
5. Good friends. Especially good friends who get why we sometimes have to bail on plans to salvage an experiment. Especially good friends who understand the ups and downs of research and academia. Especially good friends with an ice cream maker.
6. Our critters. They are manic and irrepressible. They make us laugh on a daily basis. It's good to be responsible for caring for something outside of one's self. It helps to maintain perspective.
7. My new pie plate. I got this one from Mississippi Mud Pottery on Etsy. It's beautiful and it has a hook on the back so you can hang it on your wall as art! Also, you can make pies in it. If you're very lucky and I'm very good, I will post pictures of a peach pie in the near future.
8. In spite of recent setbacks, I am still going to graduate this fall. This is a Very Good Thing. Recent setbacks haven't really cast aspersions on this goal, I'm just reminding myself that things are still heading in that direction. Don't sweat the small stuff.
9. I've got several projects on the go right now. Sometimes this feels overwhelming, but honestly, I do my best work when I've got several balls in the air. I've got a fantastic undergrad minion that I've trained up to be a second pair of hands, and he really has increased my efficiency so that's one project that keeps plugging on. Another is with a collaborator who is just a remarkably good person. He has been very understanding about my priorities with respect to this project and encouraging about my other projects with which he is not even involved. Cool guy. I really love working with him and I'm excited about where this project is headed.
10. My Better Half. He puts up with my rages over experimental failures, and still seems to like me. He's been through the Ph.D. wringer himself, so he knows I won't be this nuts forever. He's an adventurous eater (and an excellent cook himself), so I get to try out new stuff in the kitchen (squid tonight dear?). Mostly, he makes me laugh and keeps me sane. I really like that.

You have got to be kidding me [UPDATED]

I was planning on taking images for a ManuBeast figure today, which meant a whole day's worth of prep. In between steps I was setting up for a second batch tomorrow. I just tried to image the first batch.

The laser is fucking dead.

Of course the fucking laser is fucking dead. How could it be otherwise on the weekend that AA forfeits for the sake of data collection (and because the scope is booked solid all week)?

First I thought I fucked up - that I must have screwed something up in my protocol, but it's the fucking nuclear counterstain. There is no way to screw it up. Then someone told me that somebody else had the same fucking problem yesterday, but didn't say anything to the other users or to the person in charge of fixing shit. WTF!!!!!?!??!?!? Why would you not tell anyone that the fucking laser was dead on a weekday when someone could have fixed it!?!??!

Sonofabitch!

I could have spent my day making ice cream and peach pie. Instead I was here prepping 25 fucking samples for imaging that amounted to exactly zero. And I've started another 25 fucking samples for tomorrow that will also be a total waste. It's a good thing that nobody else is here today. There's a good chance I would be cracking their heads.

For the love of all that is good and right in the universe, why can't I just get these last fucking pieces of data so that I can submit this godforsaken paper!?!?!?!?

[UPDATE]: Came in on Sunday to finish samples that I started yesterday. Just for shits and giggles, fired up the scope. It is clear that someone's been at the scope since I was yesterday, though no one is signed up, and fix-it dude most certainly will not be in on the weekend. Hmmm. Strangely, I now get signal from my nuclear counterstain (HOORAY!)...but only on 1 out of 25 slides, and on the "positive control" slides that I did last week (WTF!). I checked all of these slides yesterday, no signal on ANY of them. So the good news is that the laser is not dead (today), but the bad news is that something is clearly wrong with my nuclear stain. None of these samples are going to give me publication-quality images now, so that's a total of 50 samples and three days down the drain. Now that I've got my immnuos working (no trouble with counterstain during that ordeal) I have to troubleshoot the nuclear counterstain??? You're joking right??? Why is it that this has all worked for me for the last 4 years and the sky starts falling in now, when I really need to just snap some pretty pictures and submit the fucking paper? Is the universe conspiring against me? Am I not supposed to graduate? Now I just feel like I'm losing it. I am totally convinced that the laser was dead yesterday, but it is working today. Am I crazy?? And what the hell with the nuclear stain?? I have not changed a damn thing with the protocol, and the slides I stained last week have beautiful nuclear signal...am I cursed?]