Oxford – Bring on the Thesis

I’ve been back in Oxford for two weeks now.  Finally got over my jet lag. Maybe I shouldn’t have slept during the daytime the entire flight over from the States. But it took me longer than usual to get over the jetlag – in part due to a newfound love of afternoon coffee and partly due to what is possibly the best duvet in the world.

 

So how’s the thesis writing going, you ask? Well, I must say that the Radcliffe Camera in Oxford is an amazing place to work. Opened in 1749, it’s a place full of history, and the sound of students working. There are also many coffee places I go to when in need for more ambient noise, and caffeine.

 

But the writing, I hear you ask, how is the writing going? Well, the first thing to do is to figure out the data that you have. To this end I need to finish analyzing some data, which is what I’m currently focusing on. Also, for the writing part, I’m working on the words that go in the results section. The Results section is always trickier than it might appear because all the figures and relevant graphs need to appear there in order to present the data, but in terms of prose it’s important to provide context for the figures, while at the same time not including that which should instead belong in either the Methods or Discussion sections.  Often, so far, I’ve found myself writing and then thinking that a paragraph really belongs in another location in the thesis.

 

So here are the steps now:

1)     Finish analyzing data

2)     Write Results section

3)     Make figures and insert into Results section

4)     Reformat Methods and Intro to fit around the Results section

Stuck in a Hurricane – Don’t Know When I’ll Be Back Again!

Hurricane Sandy will go down in history for what it did to New York and New Jersey. But it will also be a story I will tell my grandchildren.

 

It’s stressful enough to move out of an apartment.  You have to sell or give away all of your furniture. You have to throw out all the detritus of a private life that you would keep using if you were still there – but which everyone else already seems to possess and doesn’t want more of: stainless steel pots anyone?  You have to clean the apartment thoroughly if you expect any of your deposit back. And you have to pack your entire life into 2 check-in bags (I paid extra and brought 3), one computer bag, and one “personal item”.

 

It’s also stressful to finish your PhD experiments (see my previous post: “Experiments – Done!!”). To simply circle a date on a calendar and say, “I’m going to finish this day and what I’ve got is what I’ve got. It is, what it is, and I’m going to write up and be done.” Particularly stressful when it gets to 3 days before you leave and you’re starting an 18 hour experiment and still have immunohistochemistry to work out, and to make sure you have all your files off all the various computers in the department.

 

And then: Hurricane Sandy. Oh, how I love you Hurricane Sandy. I’ve seen stronger wind and rain growing up in Albuquerque; and yet, for some reason, Washington D.C. completely shut down. I had just gotten back from my birthday/going away party on Sunday night, when they announced that the metro was going to be shut all day Monday. I therefore had a choice: I could either be stuck in the apartment with no way to get into work, or I could be stuck at work, with no way to get back to my apartment.  The choice was simple, I had an experiment that had to run overnight Monday to Tuesday. Either that experiment was going to be in my thesis or it was not. Also, my immunohistochemistry wasn’t working yet. I had to go into lab. So, I grabbed a set of pajamas, and hoofed it into lab.

 

I spent two nights in the lab.

The experiments got done.

The files were obtained.

The immunohistochemistry finally worked.

I ate the cheese, salami, and cracker platter leftover from the Friday birthday party from the lab fridge. I ate leftover pie.

Did I mention I spent my birthday in the lab with a hurricane outside?

 

Then I went home.

I packed everything I could.

I threw out everything I couldn’t.

I scrubbed every appliance and floor in the apartment.

I slept one night on an air mattress (a step up from the chairs in the lab).

Then I went to the airport and flew to England.

Experiments – Done!! a.k.a. I Never Want to Do Immunohistochemistry Again

Those who have been following me the past month on Twitter have witnessed my countdown to leaving the NIH. For those reading my blog, apologies for not posting, but time was limited and there was a lot of work to be done. So I now post a recap:

 

I needed to make sure I had all the information necessary to create a coherent project. To this end, the main focus of the last month was making sure I had all the staining I needed from my transplants in order to correlate angiogenesis and good bone formation, so that I could justify my thesis.

 

To this end, a preliminary survey of all of the “good transplants” – ones that I had transplanted since my return to the states in 2010 was made. This meant that 180 slides were observed in H&E. It was found that a number of transplants were in fact mammary glands or plain brown fat. The explanation for this is that sometimes transplants are resorbed – one of the reasons, after all, why I focused on collagen-based scaffolds is because they are resorbed over time. However, the ideal would be for the scaffold to be resorbed but simultaneously replaced by bone. Since this does not always happen, I was occasionally hunting around in the mouse for anything that resembled a transplant. After the preliminary survey, samples that were not transplants, or which were transplants but for reasons unknown were very fragmented and deemed “impossible to section” were abandoned. This left about 130 samples.

 

Some of the remaining samples were re-stained, and all were scanned using an Aperio slide scanner. This is an amazing machine, which is the saving grace for someone like me – a graduate student who vehemently despises microscopy. Slides are scanned at 40x resolution and then the entire image can be viewed on a computer, zoomed, and images captured of any section desired – without having to constantly refer back to the actual slide. Also very handy for me because it meant all I had to bring to Oxford was my computer. Welcome to the digital age of science.

 

Finally, all of the 130 slides to be analyzed were graded for bone formation and hematopoiesis (marrow formation) on a scale of 1-4 by 3 “blinded” researchers. I use blinded in quotation marks here, since the identities of what the samples were clearly written on the slide, but I have a habit of using acronyms for anything, so no one knew what they were. As one of the “blinded” researchers myself, I simply didn’t look at the slides based on identities, but by their numbered position in the box – and, as they were not arranged in the most logical order, my gradings can also be seen as unbiased.

 

Some of the slides appeared to be either pre-osteoid (i.e. in the process of forming bone) or cartilage, so I stained with toluidine blue. However, none of these samples turned out to be cartilage. For grading purposes these samples were deemed unsuccessful.

 

Then, I came to what I had been dreading for months. Immunohistochemistry.

 

Often shortened to IHC, immunohistochemistry is simply the staining of sections of tissue for specific expression of markers through the use of antibodies. Sounds simple, but not all antibodies are created equal and it took me about 10 different antibodies to find one that was specific for blood vessels and worked on my sample. Adding to the complexity of this process is the need for heat retrieval of some markers – for which every company and scientist will tell you a different protocol. Yet another variable exists in how to show where you have specifically selected with your antibody (i.e. choice of a secondary antibody). Since most antibodies aren’t themselves fluorescent or colorful (chromogenic) this means that you have to create another layer of antibody by selecting for the first one with your secondary which is either fluorescent or colorful. Thus, I think it’s easy to see that what, in theory, could be a very simple process, is indeed a very complex one.

 

But, I got it to work. I finally got really good immunohistochemistry of blood vessels on 16 representative samples from one experiment. The rest of the samples I am going to have to attempt to count the blood vessels by eye based off the H&E staining scans that I have.

 

And then there was the hurricane. But that’s for my next post!