Debbie Knight

Archive for the ‘research log’ Category

A Day in the Life: March 7, 2013

In A Day in the Life, research log on March 7, 2013 at 4:56 pm

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab. These entries I will call “A Day in the Life…” 

Today I’m searching for an antibody that I can use to stain the cells that line blood vessels (endothelial cells) in mouse tissue.

And I want to visualize where the antibody binds in that tissue with a dye that glows (or fluoresces) under certain conditions.  Something like this:

MEK14.7 ab8158_3

From abcam.com: Ab8158 at a dilution of 1/50 staining CD34 from mouse lung cells by Immunocytochemistry

A Google search has turned up several results that look promising.

However, there’s some cross checking that has to happen before my lab will plunk down $300 for the antibody. Sure, I could take the company’s word that it works in fluorescent staining techniques, but I need more proof. I need to see that it will work in my experiment and that means finding published images in the scientific literature.

Seeing, after all, is believing.

This process can take time.

For the antibody I’m looking for, there are at least three different versions (or clones).

I’ve spent a couple of hours trying to find just the right one, sifting through the “data.”

The antibody I’ve chosen, clone MEK14.7, seems to work in just about any experimental application. It’s rare to find an antibody that works in fresh samples as well as formalin-fixed samples, but this one does. Why? The molecule the antibody binds to in the tissue (called an antigen) often changes shape when fixed in formalin. The shape determines if the antibody can bind. If it changes, the antibody no longer recognizes (and binds) it.

Now, if I can find this antibody with the right dye on it, life will be good.

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Update

If you look at the comments to this post, you will notice that Tom suggested the website 1DegreeBio. com  

I was unaware of this website, but it would have saved me some time in searching for an antibody.

I’m pretty sure I would try it first for any of my antibody searches.

The only thing lacking at this time (and it will improve over time as visitors add their two cents) is there were no ratings/reviews of the antibody reagents I looked at. This would be a big help, although I would caution that if the reviews are done by individual researchers, they should be taken with a grain of salt:  what works well in one researcher’s hands may not in the hands of another.

Why? It could be how the sample was handled before staining with the antibody (such as storage or fixation). It could be the incubation time of the antibody with the tissue — some labs do an overnight incubation in the cold, others (such as myself) incubate an hour or two at room temperature, and some incubate for 30 minutes at body temperature (37 degrees Celsius). These are only two examples, there are several steps in the staining process that could influence the antibody’s performance in an assay. 

Thanks for the suggestion, Tom! 🙂

A Day in the Life: January 28, 2013

In A Day in the Life on January 28, 2013 at 11:54 am

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab. These entries I will call “A Day in the Life…” 

This week I am making thin slices (called sections) of frozen tissue specimens using an instrument called a “cryostat.”

This is a cryostat, an instrument used to make thin slices (sections) of frozen tissue samples.

This is a cryostat, an instrument used to make thin slices (sections) of frozen tissue samples.

The one I’m using is a little fancier than the one I used ten years ago. This one has a digital display and controls that you can adjust.

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This cryostat’s controls are digital, so fine adjustments can be made easily.

The inside, however, looks pretty much the same as the “old” cryostat I used to use.

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Inside the cryostat.

So, the first thing you need is your frozen block of tissue. The “block” is formed in a plastic mold called a “cryomold” which can come in several sizes. Basically, you put your tissue into the mold and then pour this clear gooey (think honey) liquid called “O.C.T embedding medium.”

OCT-medium-sm

The all important O.C.T. embedding medium is a clear and gooey substance that turns white when frozen.

You then quickly freeze (called “snap freeze” or “flash freeze”) the block in a really cold liquid like liquid nitrogen or dry ice-chilled ethanol or isopentane. The clear liquid turns white that’s seen in the photo below.

cryomold-sm

Tissue (red) flash frozen in O.C.T. embedding medium (white) in a plastic mold.

We store our embedded tissue blocks at minus 80 degrees Celsius until we are ready to section them.  The tissue block is pretty brittle at this temperature so we have to “warm” it up to minus 27 degrees Celsius (the temperature inside the cryostat) before we can proceed. You can do this by placing the tissue blocks inside the cryostat and waiting 15 to 30 minutes before proceeding.

Once the tissue block has warmed up a little, you “pop” the tissue block out of the plastic mold. You then add some of the O.C.T. embedding medium to the cryostat adapter (called a “chuck”) and quickly place the tissue block on top of it. The embedding medium acts like a glue to hold the block onto the chuck.

tissue-on-chuck-sm

To attach the frozen tissue block to the cryostat adapter (called a “chuck”), you add a few drops of O.C.T. embedding medium (as shown on right) and quickly place the tissue block over it to essentially glue the block on the chuck (as shown on the right).

Here is the result. I obviously am a little out of practice getting the tissue on the chuck straight, but you get the idea.

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Tissue block is mounted on the cryostat chuck. In the background, other tissue blocks are awaiting the same fate.

The next step is to place the chuck on the mount.

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Placing the chuck on the mount of he cryostat.

The chuck/tissue block is adjusted so it is as close to parallel with the cutting blade as possible. It is then locked into place to prevent it from shifting while sections are cut.

chuck-3-sm

Adjusting and locking the chuck/tissue block into place.

The cutting surface is a blade that sort of resembles a razor blade. It comes in a dispenser pack as shown in the photo below.

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A pack of blades used to cut tissue in the cryostat. They’re kind of like razor blades.  Only one blade is used at a time.

The blade is placed in the holder and locked down. This blade will be used over and over until it has too many nicks or the cutting edge or becomes dull. It is really sharp, so the user has to be careful not to accidentally cut herself on the blade. Some cryostats are used to cut potentially biohazardous materials like human tissues or infectious animal tissues.

blade-on-microtome-sm

The cryostat blade goes here (indicated by pointing finger). It’s really sharp, so care is needed when working with the cryostat.

To cut tissue sections, you use a hand crank on the outside of the cryostat to move the tissue across the blade. It’s nice because you can control the speed of the cut — fast if you’re trying to get to a specific area of tissue or slow if you’re planning on catching and keeping the section.

To make the chuck/tissue block move across the blade, you turn a wheel on the side of the cryostat. You can turn the handle slowly for more precision work or quickly to trim the block to an area of interest.

To make the chuck/tissue block move across the blade, you turn a wheel on the side of the cryostat. You can turn the handle slowly for more precision work or quickly to trim the block to an area of interest.

cutting-tissue-composite-sm

The chuck/tissue block moves across the blade as shown in this sequence of photos.

This particular cryostat is missing a part: a thing called a “roll plate.” The roll plate is a flat piece of plastic that rests against the blade and catches the tissue section as it comes off. It prevents the tissue section from rolling up like a scroll. In lieu of a roll plate, you have to use a paintbrush to catch and unroll the section as it comes off the blade.

catching-the-section-sm

This cryostat is lacking a device called a “roll plate” which helps to catch the tissue section and lay it flat before it rolls up into a tube. Here I am using a camel hair paint brush to catch the tissue section.

The microscope slides used to catch the sections have a special coating on them to help the tissue “stick” better (and stay stuck) to the glass.

slides-sm

The microscope slides we use have a special coating on them which helps the tissue “stick” better to the glass.

Once you have flattened out the tissue section with the paintbrush, you then touch the glass slide (face down) on the tissue. The glass slide is room temperature, the tissue section is cold (at minus 27 degrees Celsius), so the tissue section “melts” onto the slide. You then let the section air dry at room temperature before placing it in a slide box that will go into a freezer for storage.

transfering-the-section-sm

After the tissue section is laid flat using the paintbrush(es), you tap the glass slide (face down) against the tissue section. The section sort of melts onto the slide.

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The tissue needs to dry a little after it is transferred to the slide.

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The slides are placed in a slide box. The lid is put in place before the slide box goes into the minus 80 degree Celsius freezer for storage.

Not every tissue section that rolls off the blade is usable. Sometimes you just can’t unroll it. Sometimes the sections tears. After a few hours of cutting tissues, you end up with a nice pile of shavings. These have to be cleaned up and disposed of properly.

leftover-sections-sm

Not every section is successfully uncurled. Here are some discarded sections.

So there you have it.

And even though it’s been a good ten years since I last used a cryostat, I think it’s a lot like riding a bike. You might be a little wobbly, but it all comes back to you.

A Day in the Life: November 14, 2012

In A Day in the Life, research log on November 14, 2012 at 3:51 pm

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab. These entries I will call “A Day in the Life…” 

This week I’ve been burrowing through scientific journal articles, ferreting out information that might help move my research project along. We, in the scientific community, call this a “literature search.” And my work is starting to pay off. So far, I have found one really solid lead that I may pursue.

While I was searching the scientific / medical article database called PubMed (maintained by the National Center for Biotechnology InformationU.S. National Library of Medicine), I noticed a cool feature that I hadn’t seen on this site before. There was this little chart (a histogram) that tells you how many articles were published each year on your topic of interest.

This is useful in that it tells you whether you’re looking at a “hot” area of research. Or what years the research was hot.

For example, I typed in the term “immunoglobulin and complement fixation” and saw this chart:

So in 1973 (the first bar on the left) there were 643 articles published. If I scroll back a few years, the research actually peaked in 1971 with a whopping 730 published articles.

And in 2012? (the bar on the far right) A measly 15 articles. Obviously not the hotbed of research it once was.

When I searched “immunoglobulin subclass,” I got this result:

I’m not sure what was going on in 1990, but that seems to be the peak of this topic’s publication, maxing out at 267 articles.

And when I typed in “nanoparticles” (a relatively new area of research), I got this:

Last year, there were an incredible 11,756 articles published on this topic. Talk about “hot” area of research! This dwarfs the previous two searches. And I’m sure that number will continue to climb.

I was amazed there were two articles on nanoparticles published in 1978.

I’m not sure when PubMed added this feature. Admittedly it’s been a few months since I searched for articles. But I like it.

A Day in the Life: November 2, 2012

In A Day in the Life, research log on November 2, 2012 at 12:57 pm

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab. These entries I will call “A Day in the Life…” 

Yesterday’s experiment  didn’t go so well.

And it wasn’t even really my experiment. My lab was helping out a newly-hired pathologist get some data that he could possibly use in a grant proposal.

We treated some cultured cells (specifically ones that line blood vessels called endothelial cells) with a potential cancer drug. This drug may work to treat brain tumors in two ways — directly on the tumor cells and on the blood vessels that supply nutrients and oxygen to them. We treated with several doses of the drug for different amounts of time. We stained the cells for a couple of things that would show us if the drug concentrations were toxic to the cells.

To look at those markers, we used an instrument called a  flow cytometer. This machine slurps up the cells floating in a buffer solution and marches them single file past a laser beam and a series of sensors. One of those sensors counts the cells. Ideally we would count 10,000 cells, but the samples were taking a long time to run. We settled on counting only 5,000 cells — more than enough to get valuable information from the other sensors.

Some samples took only a couple of minutes.

Not ideal, but I could live with that.

But some samples took a lot longer. And some never reached the 5,000 cell mark. For example, the cytometer had only counted 3,375 cells in six minutes and 28 seconds in this particular sample (the highest dose of the drug). Argh!

It was very frustrating.

It  was also a little boring watching the samples run.

To keep myself busy (and entertained), I read a journal article, perused the school newspaper, outlined experiments that I needed to do, doodled, took some photos with my cell phone for this blog, worked on a crossword puzzle, checked my cell phone, crunched the results that slowly trickled in, etc.  I also used the data as a Rorschach test (of sorts), trying to see if I could “see” anything in the data. 

Rorschach test? What do YOU see?

The first set of samples (of a total of six sets) took two hours to run. This was all at top speed on the cytometer, mind you. The proverbial pedal was to the metal.

It was then I made an “executive decision” to run only two of the three samples per treatment. This, I hoped, would give me adequate data.  And I wouldn’t be running my samples, one at a time, manually for 12 hours straight.

It only took half that time!

Six.

Long.

Hours.

No lunch. No break.

Sometimes that is a day in my life.

A Day in the Life: September 27, 2012

In A Day in the Life on September 27, 2012 at 2:59 pm

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab. These entries I will call “A Day in the Life…” 

Yesterday we got word that our grant proposal did not get funded.

Sad news for us, especially since this was the second (and final) time the proposal was submitted.

But with current pay lines at something like the 6 percentile, most grant proposals do not get funded.

I think what stung the most was the proposal was not even scored.

This means that the two or three scientists who were assigned to thoroughly read and evaluate the proposal did not think it was worth further consideration by the other scientists in the panel. The panel is often called a study section.

While it is technically a collective decision, the assigned reviewers wield quite a bit of sway because they have read the proposal in more depth than the rest of the scientists in the study section. So if your proposal failed to razzle-dazzle the reviewers, you don’t make the cut.

So while we thought we had a great research idea, there were scientists out there who didn’t agree.

Such is the way of research.

So, for now, it’s back to the proverbial drawing board.

And that about “covers” it

In research log on September 20, 2012 at 9:30 am

So yesterday, I posted the Photo of the Week of a lab chair enshrouded in black plastic and green labeling tape. A pragmatic way to address lab safety when working with biohazardous materials. Specifically, no fabric-covered chairs are allowed in a biosafety level 2 designated laboratory.

My lab had two chairs, used in a desk area, that were fabric covered. Something we finally had to address after a lab safety inspection.

The chairs in question were great lab chairs. We didn’t want to cover them in a black plastic trash bag secured with duct tape.

I considered clear plastic vinyl — you know, the stuff with which some people cover their sofas. But when I went to the fabric store, the fine print said that this was flammable. Not something that we would want in the lab where safety is a huge concern.

I did find some non-flammable black vinyl that I thought would work. I bought a yard for $16.99  which turned out to be just enough to cover the two lab chairs.

I should note here that this was only my second attempt at reupholstering and I didn’t do anything “fancy” here.

The learning curve was actually figuring out how to remove the seat forms from the chair. The rest was pretty easy.

First step was to remove the seat bottom and seat back from the lab chair.

The next step was to make a “pattern” to guide how I would cut the vinyl.

Next, I gently “stretched” the vinyl over the seat back form, using a staple gun to hold in place.

Then, I stapled the fabric all around the seat back form. This material wasn’t going anywhere when I was done. Not pretty, but no one will see it (well, except you).

The final product. I’m not entirely happy with the corners, but the vinyl reupholstery looks much better than a black plastic trash bag.

Two lab chairs, reupholstered for less than $20.

They may not look quite as nice as they did in their former fabric-covered selves, but at least they are safe to use anywhere in the lab now.

Sometimes you have to get creative in the lab

In research log on September 12, 2012 at 9:30 am

Sometimes you have to find creative solutions when you do an experiment.

I was running a gel on a much larger format than I am used to. It felt like I was using a gel held between two glass plates that I imagined were made for the Jolly Green Giant.

I typically work with the smaller glass plate (between my thumb and index finger), but today I was working with the larger glass plate.

I usually run the gels in the smaller apparatus on the left, but on this day, I was using the ginormous apparatus on the right where I encountered several logistical issues.

Because I had never worked in this scale before, it meant I encountered several logistical issues as I conducted the experiment. Most I could solve pretty easily. For instance, I never run gels in this area of the lab, but I needed a steady stream of water to cool the buffer. I had to figure out where to safely put the power supply where it wouldn’t get wet should the setup spring a leak (think tubing popping off the connector and spraying water everywhere). Also, I had to figure out how to pry the gel from between the glass plates without tearing it. Little things like this.

But the biggest problem I encountered was how I would incubate the resulting membrane strips for the rest of the experiment.

I thought I could perhaps place them in this tray designed to hold thin strips of membranes. However, my strips were twice as wide as the slots in the tray.

The tray I thought I might be able to incubate strips of membrane for my experiment. The strips were too wide for the slots.

I dug through our cabinets and drawers and found four screwtop glass tubes that would work. The problem was there were only four — I needed 20.

Problem: I only had four screwtop glass tubes to work with — I needed 20 of them for the experiment.

It was a holiday. I couldn’t turn to my lab neighbors to see if they had any of these test tubes.

So, I had to get creative.

There was nothing in the lab that would work. So while the gel was running, I went to a couple of nearby retail stores. I kept an extremely open mind, but I didn’t find anything that would work.

Hmmm.

I next tried a hardware store. Again, open minded. I finally found some PVC pipe and end caps that could work. The hardware guy didn’t bat an eye when I said I needed 20 six-inch pieces cut from a ten-foot pipe.

Twenty PVC “tubes” that the hardware employee had to cut from a ten-foot pipe.

So, I had my solution. It wasn’t ideal — you can’t exactly see through PVC pipe. And getting the caps off wasn’t easy. In fact, I didn’t have quite enough caps, so I had to use rubber stoppers for some of them — turned out the stoppers were much easier to remove when I had to add reagents to the “tube.” But it helped me get through the experiment.

The PVC pipe solution.

It should be noted that the PVC “tubes” cost about 50 cents a piece. The glass tubes from a lab supplier cost two dollars a piece.

Oh, I did end up using the tray — to keep the tubes in position while they rocked overnight.

To keep the “tubes” in a specific orientation, I used the immunoblot tray. The slots held everything in place pretty well.

In the research lab, it often takes some creative thinking beyond designing the experiment — sometimes you need it just to do the experiment.

I guess that’s why there’s the saying “necessity is the mother of invention.”

A Day in the Life: August 30, 2012

In A Day in the Life, research log on August 30, 2012 at 9:30 am

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab. These entries I will call “A Day in the Life…” 

We have a film processor in our lab. We use it to develop plastic films that have been exposed to small amounts of light produced by a chemical reaction to help us look at proteins in our samples.

The film processor uses three solutions: a developer, a fixer, and water.

Usually our developer and fixer solutions are delivered by our service guy in a carboy.

However, we ran out of developer and they shipped us a bunch of smaller bottles. I thought it was a little weird that there were two different sized bottles in the box. It wasn’t until I read the directions on the back of one of those bottles that I realized that some assembly was required.

This was a first for me!

One bottle of solution “A” and one of “B” and an equal amount of water.

Sounds easy enough.

And it was pretty easy, but I think I prefer the service guy bringing it already made. Less time, less mess.

A Day in the Life: August 23, 2012

In A Day in the Life, research log on August 23, 2012 at 4:50 pm

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab. These entries I will call “A Day in the Life…” 

The past two days I’ve been busy wearing a “catcher’s mitt.”

A newly-hired faculty member shipped his irreplaceable research specimens from California to me. He shipped them in two overnight shipments on dry ice. The dry ice was to keep the samples frozen during their journey.

He sent the samples in all 15 styrofoam boxes shown in the photo. This kept me busy as I had to unpack and transfer everything in these boxes to the -80C freezer — without getting frost bite, I might add.

While everything arrived in great condition and the packages could easily be tracked on the Internet, he still worried about them. I received phone calls and emails from him before and after the packages arrived. First to give me a head’s up the samples were on their way. And then, after the packages arrived,  asking if the samples were okay and reminding me yet again where to store them.

I guess I can’t fault him. The samples are, after all, irreplaceable. I suppose I would have done the same if the roles were reversed.

Day in the Life: August 10, 2012

In A Day in the Life, research log on August 10, 2012 at 9:00 am

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab on a particular day. These entries I will call “A Day in the Life…” 

My division has hired two new neuropathologists. One will arrive in September and the other in October. There has been quite a bit to do in preparation of their arrival. Politics, “luck,” or whatever you’d like to call it, I’ve had the honor of doing some of the prep work.

One task I had to do: clean out a freezer that belonged to a researcher turned administrator who has since passed away.

My job was made easier when I discovered that the minus eighty degree Celsius freezer had warmed to a balmy minus four degrees Celsius. Not great for the stuff in the freezer — in fact what alerted me to the problem was that some samples were liquid when they should be frozen solid.  But is was great for my fingers which were no longer in danger of getting frost bitten.

This is what the freezer looked like before I really started. A “good” example of how not to store stuff in a freezer, with boxes of stuff stashed all willy-nilly.

And this was after the clean out.

In many of the boxes, cultured cells were stored. Not the best storage conditions for cells. Ideally, cells should be stored in liquid nitrogen or a -140 freezer. The cultured cells I had to treat as if they were biohazardous material. So I had to put them in the appropriate waste container.

As I was dumping the vials of cells out of the cardboard boxes, I thought about all the time, effort and money that had gone into growing the cells  and freezing them. And how much time was taken just labeling the darn vials. Let alone the work, sweat and tears that went into the many experiments and experimental reagents also stored in this freezer.

It was sad to see a lifetime of research efforts going into the waste bins and boxes.

And labels on boxes show the many people that worked on those experiments. Take one box, clearly important to research by what Ms. Boardman wrote on it: “Do not use. All pCB original clones. Original DNA from which all other preps have been propagated.”

Of course, some boxes, though labeled, were not as informative. One box was labeled “Unknowns.”  This was box “A” implying there were more unknown samples lurking in the freezer.  I  did not find this label very reassuring. I’m not sure why one would keep samples that were an unknown entity. Perplexed, I placed in the biohazard box just to be sure.

In addition to biological specimens, there were bottles of chemicals that I had to set aside for proper disposal by the university’s Environmental Health and Safety personnel.

There were volumetric flasks full of unknown solvents. This lab was known for its work in brain lipids — difficult to work with and to extract. These were also set aside for the EHS people to determine proper disposal.

The oldest documented chemical in the freezer? It was an enzyme from 1988, based on this paperwork I found with the vial.

Although I did find a really old canister of cholesterol that might have been older. But with no date on the label and no documentation to back it up, I couldn’t really say.

There were some reagents from familiar suppliers but with unfamiliar labels (meaning the chemicals were pretty old).  The DH5-alpha cells in the photo below are still sold by Life Technologies, but I certainly don’t remember Bethesda Research Laboratories.

And, in all this excavation I did happen to find the weirdest thing I have ever found in a freezer, though it shouldn’t surprise me since this was a neuropathologist’s freezer.

A slice of human brain in a box.

It was a little freezer burned, as you can see.

I’m sure the person’s last thoughts had not been: “I wonder if someone will find a slice of my brain in a freezer some day …”

So I will end my archaeological forays into the frozen tundra of a researcher’s freezer here.

But I will ask the question to the researchers out there: What is the weirdest thing you have found in a laboratory freezer?