Week 2: Guts and guts, awaiting glory

Mar 29, 2019

As the title so subtly suggests, this week did, in fact, include some operations with intestinal organs.

First thing on Monday, however, I had to do some control tissue optimization building on last week’s slides, in order to adequately be able to interpret the initial tests. A fidgety microscope did not help, but once the slides were stained and all was sorted out I got back to the slides on Tuesday. Some of the mice needed to be administered medications to, but since they did not want to take it through common oral means, it, unfortunately, required gavaging.

I spent the whole of the day on Tuesday in the dark microscopy room taking pictures of the slides from last week and Monday. Though this task is time-consuming, tedious and most definitely not enjoyable, I found that a good cup of coffee and some nice tunes in my ears makes it bearable at least. Though the slide numbers are randomized so that researchers are “blind” to ensure that they do not have a bias, I began to notice interesting patterns by the end of the day. Though I was still not finished taking pictures of all the slides, I decided that I would spread out finishing the rest of them over the remainder of the week.

On Wednesday, in addition to culturing and splitting some overgrowing cells, we decided that we should try to approach the experiment from a different angle as well. A protein-specific quantizing leading to a western blot, distributing the proteins through electrophoresis by their respective molecular weights, would help me in the analysis of protein concentrations, and so since the small and large intestines of the eight mice were saved and stored in a freezer, I only needed a small piece of each of the sixteen samples. Locating them and allowing them to thaw, I tried to cut small pieces of the extremely slimy and slithery guts since I only needed enough to make protein lysates, and would still perhaps need to use the same samples again later.

Continuing the process to make protein lysates for the western blot on Thursday, giving a mere three microliters of protein lysate to a technician, the next day, Friday, he was able to give me a critical table with information regarding the concentration of proteins in each sample. This would help to adjust the samples by diluting them so that they would be of consistent concentrations, making for a plausible reading of the two blots.

Friday was spent preparing and running the gels. Since much of this is done in time chunks of an hour or so, I had time to take more tissue pictures in between the western blot and gel procedure. By the end of the day, I had finished taking pictures of all the skin and small intestine samples, with only the large intestines remaining, which would have to wait for next week to be completed. After the gels were finished running, and I transferred them to a special tissue paper, I stained them and left them in a milk blocking buffer solution for some time after which they were stored in a freezer so that I could finish taking the western blots on Monday.

This is a picture of skin tissue I took where we are looking for CD4s that are stained in brown. You could see them mostly congregating around the hair follicles.

 

This is a large intestine tissue sample

 

Running electrophoresis on a gel for a western blot

 

WB after stain prior to imaging

 

 

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