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Following on from the first part of the H and E 101 articles, here are the materials and recipes you’ll need for your own H and E workstation (assuming you don’t have access to a histology lab). Many of the chemicals listed below are toxic and/or harmful. Use PPE when handling/storing, follow SOP’s in your…
The Irish Famine (or ‘Great Potato Famine’ if you live outside the Emerald Isle) killed one million people and forced another million to leave the country between 1845 and 1852. It was caused by a blight on the country’s main food stock- the Irish ‘Lumper’ potato. Now, researchers have identified the genome of the blight…
Haematoxylin and Eosin staining is the most common staining in the modern (and old!) histology lab. This staining technique gives an overview of the structure of the tissue and can be used in pathological diagnosis. This article follows on from Nicola’s introduction, but we’ll take an in-depth look at the stains, chemistry and method to…
I was first introduced to Conrad Waddington’s epigenetic landscape when reading ‘The epigenetic revolution’, a fantastic introduction to epigenetics, and in my opinion, a must read for anyone who is looking for an entertaining and enjoyable introduction to this fascinating field. In his model, Waddington likens the process of cellular differentiation to a marble, which…
Unlike immortalized cells lines, primary cells can only be kept in cell culture for a finite period of time, if at all. Therefore, you often need to obtain primary cells directly from an animal source. After which, you may fix and image the primary cells in situ (as part of the whole organ), or you…
Isoelectric focusing electrophoresis (IEF) of proteins is nowhere near as popular as its cousin – sodium dodecyl sulphate-polyacrylamide gel electrophoresis aka SDS-PAGE. While in both methods the proteins are denatured, IEF is a gel-based electrophoretic separation of proteins using difference in their overall charges. The sodium dodecyl sulphate – SDS part of the usual gel…
Fixing suspension cells for imaging can be trickier than fixing adherent cells, as they can’t be cultured on a coverslip. Discover how you can stick them down with the help of centrifugation.
Wouldn’t it be great to put your nucleotide sequence into a program and get back a 3D-structure of your protein and a full description of its functions? In theory, because the protein 3D-structure is determined by the aminoacid sequence, given the right algorithm and a powerful enough computer, this should be simple. In practice, because…
You just can’t put raw tissue or cell samples on your slides and expect good histology results! Instead you must preserve or ‘fix’ your samples. Fixing ensures that your cell structures stay intact and that your antigens are immobilized. Ideally, fixation would also still permit unfettered access of your antibodies to your antigens. However, as…
According to the central dogma of molecular biology, DNA is transcribed into RNA, that is translated to proteins. Inconveniently, the vast majority of the genome contains sequences that do not actually code for proteins. So, this non-coding RNA (ncRNA) was dismissed as non-functional junk, letting researchers tick the box on their to-do lists and head off…
The theory behind the idea of having shared microscopes is a good one, but, in reality, this can sometimes mean you have to put up with the dirty habits of your fellow scientists and researchers. And some of your lab mates turn out to be really mucky! Here’s my Top 10 of things which really…
Recently BsB author Yevgeniy Grigoryev shared a total RNA isolation protocol. The one I use is even simpler—no expensive Trizol, which is a mix of phenol and some salts, all that is required is some Tris, SDS and phenol/chloroform mix. I have never used this protocol on non-yeast cells but I am almost sure that…
It is the black death of cell culture. Scientists don’t dare utter its name and many a graduate student has fallen victim to its indiscriminate menace. These stealthy anarchists infiltrate quietly but deliberately until their numbers swell and then they attack in strength, overwhelming their victims before they can put up a fight! What is…
Have you ever emerged from the lab, bleary-eyed, blinking dazedly at the sun after spending hours hunched over a lab bench counting endless bacterial colonies or viral plaques? A necessary evil… I consider colony/plaque counting one of the necessary evils of working with microorganisms. Necessary because many experiments have an endpoint that requires determining the…
“Do we use a monolayer or 3-D cell culture for our experiments?” A simple, yet puzzling question asked by my group leader while I was working on a drug development project at the University of Abertay Dundee. How do you answer such a question? Just read on to find out! Monolayer vs spheroid One of…
The field of epigenetics is exploding and given the strong links between epigenetic state and disease, the need to study markers like DNA methylation in humans is very relevant. This article outlines some of the main factors you should be taking into account in your study of DNA methylation in human tissues. Here goes: Biological…
Did you know fixation can mask antigen sites in your sample? Discover how you can unmask them and get your signal back on track!
Whole genome sequencing (WGS) is becoming increasingly common. Doctors now routinely order it for patients with puzzling diseases. The NHS (National Health Service in the UK) has declared that it will sequence 100,000 genomes over the next few years. Increase WGS…increase ethical questions The direct-to-consumer company 23andme has been experimenting with whole exome sequencing (WES), and another company, DNA…
To answer some of the more interesting research questions, you often need to get a good look at what’s going on inside the cell. Whether you’re running a Western blot or measuring enzyme activity, many assays require access to the materials (e.g. proteins, DNA, subcellular fragments) contained within the cell walls. There are several ways…
As part of my job ensuring plasmid quality at Addgene, I analyze 50-100 sequencing reactions a week. So I have developed some good habits that I wanted to pass on to you to make sure you are getting the most out of the data you get back from your sequencing runs. The most important of…
You’ve been told that maintaining a sterile environment in a tissue culture hood is vital to preventing contamination of cell cultures. But what exactly is meant by sterile? The definition of sterile is ‘completely clean, sanitized, and free of all forms of life’. Obviously you still want your cells and/or any other organisms you are…
Have you ever isolated a great little population of cells after days or months of trying, got truly excited about doing some immunofluorescence with them only to find out (at the very end) that all your cells washed away?! If this has happened to you, then look no further; we will introduce you to some…
Most ‘wet lab’ biologists do not have much computer programming experience, which can make downstream analysis of next generation sequencing results a bit daunting. After the sequencing platform spits out your data, what do you do with it? That’s where Galaxy comes in. What is Galaxy? Galaxy is a bioinformatics workflow management system, created by collaboration…
Unlike DNA, which can last for eons, RNA is a fragile and degradation-prone cousin. After working with RNA for a while, one becomes quite paranoid about handling RNA because even a single sneeze or drop of saliva can potentially affect your results. The reason is that there are enzymes called RNases that specifically target and…
In the previous article on EC numbers, I explained how the Enzyme Commission names enzymes, and why it is so important. In this article I’d like to take you on a brief journey through the history of the Enzyme Commission. Like many histories in science (e.g. this!), it is fascinating and gives a useful perspective…
As a biologist you will no doubt have seen Enzyme Commission (EC) numbers. An EC number is group of four numbers separated by periods in papers discussing enzymes…something like this: EC 1.1.2.1. But do you know what these numbers mean? Or where they came from? Or why we use them? If not, I will aim…
So having read our article on how a cytometer works, surely the next question is ‘what’s the right flow cytometer for me?!’ Basic Components of Flow Cytometers We know that at their most basic level, cytometers are made up of 3 main components: So, what are the considerations when selecting a flow cytometer? What…
Next-generation sequencing (NGS) really has taken the world by storm! In NGS, millions of short ‘read’s are sequenced in a short space of time, leaving you with vast amounts of data to analyze! For all NGS platforms, the input sample (i.e. your cell free DNA) must be cleaved into short sections or fragments prior to…
If you found our previous section on super resolution interesting, you may be curious for a more detailed explanation behind some of the techniques. Introduction to this counterintuitive method Of the super-resolution microscopy techniques, structured illumination microscopy (SIM) is arguably the most counterintuitive to grasp. Of course, that’s what makes it so much fun! To understand how…
“Any sufficiently advanced technology is indistinguishable from magic.” – Arthur C. Clarke In the fast-moving field of next generation sequencing, standard practices are evolving rapidly. Today, more and more labs are using Solid Phase Reversible Immobilization (SPRI) beads instead of gel purification in the preparation of libraries for sequencing. A crucial step, not for the…
Following on from our previous article, here are some suggestions for an old microscope (should you happen not to destroy it!). 1. Museum piece Start your own mini scientific instruments museum. Before you know it, you be raking through the old skips and dumpsters at your institute looking for exhibits. 2. Teach kids Teach your…
Apart from lung cells, surprisingly few cells are ever exposed to 20% oxygen, which is one of the reasons it’s hard to get in vitro cell cultures to behave. Read more about whether 20% oxygen is always the best level for cell culture and why it became the standard amount to use.
Do you see what I see? Maybe not, if the microscope is wrecked in one of these ten ways when you… 1. Carry the microscope incorrectly. A death-grip on anything but the arm and the base almost guarantees that it will slip away, crashing onto the floor to break in pieces. You don’t want a microscope which…
Did you ever encounter resistance from a mammalian cell line when trying to extract the contents? Probably not, because destroying cell membranes is easy. Cell walls, however, are a different story. They are rigid, protective layers that can be so strong that the organism gives up movement in favor of protection! Cell walls exist in…
For those of you who prepare your own DNA libraries, this article will cover the most critical aspects of library preparation to ensure a successful sequencing run. Previous Bite Size Bio articles have covered the basics of how 454 sequencing works, so give those a quick review if you are unfamiliar with the process. This video is also highly…
Do you know what your histology fixatives are really doing to your samples? Read on to learn what happens to tissue treated with two common fixatives.
What Does Oil Red O Stain? Oil Red O (‘ORO’) is used to demonstrate the presence of fat or lipids in fresh, frozen tissue sections. Introduced by French in 1926, ORO is a fat-soluble diazo dye, and is classified as one of the Sudan dyes which have been in use since the late 1800s. Like…
Want to detect iron in your samples? You need Prussian blue! Discover the incredible sensitivity of this stain and how to use it.
In my previous article on DNA gel extraction, I explained how most commercially available DNA gel extraction kits work. However, there was a time before our society was blessed with these convenient marvels of technology and scientists had to summon the gods of “Crush and Soak”. This method has been proven for millennia, as people…
Size Selection via Gel Electrophoresis Whether you are using NGS for whole genome sequencing, SNP variant analysis, HLA typing, HLA matching, or even transcriptome or miRNA analysis by RNA-seq, size selection is an extremely important consideration for optimum results. Precise size selection can increase sequencing efficiency, save money and improve genome assemblies, as well as…
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