To begin this procedure, the block of brain should be prepared for the cryostat and the section sampling frequency should be determined. Next, the antigen preserve and slides should be prepared. The brain block should be placed on the chuck inside the cryostat.
Once the brain is securely embedded on the chuck section at a preset thickness, systematic sections should be taken such that series one is placed on a slide, and further series are placed in wells prefilled with antigen preserve. Once the block has been exhausted, place the plate into a minus 20 degree Celsius freezer as part of your brain bank. Hi, I'm Dr.Mark Burke from the Visual Neurosciences Laboratory in the School of Optometry at the University of Montreal.
Hi, I'm Dr.Shahin Zur, also from the Visual Neurosciences Laboratory in the School of Optometry at the University of Montreal. Hello, I'm Dr.Mo Petto, director of the Visual Neuroscience Laboratory at the School of Optometry, university of Montreal. Today we will show you a procedure for systematically banking brain tissue in antigen preserve.
We use this procedure in our laboratory to maximize research material and as a basis for performing unbiased theology. So let's get started. This protocol describes how to systematically sample brain tissue that has been fixed, stereotactically blocked into one centimeter, thick slices and frozen.
To see these earlier steps in detail, consult our recent JoVE article dissecting the non-human primate brain in Stereotaxic space. The first step in systematic sampling is to create your sampling scheme, decide how you will section your tissue, how you will divide those sections into series or samples, and how you will arrange and keep track of those samples. First, consulting a stereotaxic atlas as needed.
Clearly define your region of interest in both the rostral, coddle, and dorsal ventral dimensions and choose your plane of sectioning. Next, decide how thick you will make your sections and calculate how many sections you can make from your region of interest. For example, from the Stereotaxic atlas, you determine that the entire ros coddle extent of your region is approximately three millimeters long.
You then decide to set the cryostat to section at 50 micrometers, and at this rate, you will have 60 coronal sections covering the entire region. At this point, you can establish your sampling interval. For a typical unbiased theological study, you need six to 10 systematically sampled sections in order to obtain reliable results.
In this example, 10 sections equally spaced throughout the Ros coddle extent result in a sampling interval of one out of six sections. Once you have determined this section sampling frequency, you should next decide which stains you want to perform. Immediately you will mount the sections you want to stain immediately, directly onto slides as you section your tissue.
For example, if you are staining with say, crestal Violet, you will capture one of six sections on a slide. This will be series one. You will store the remaining sections in plates containing antigen preserve for future immunohistochemistry.
The sampling scheme shown here consists of two slides and one standard 24 well plate of sections as part of a brain bank for a total of six series for this block of tissue. The next step is to exhaustively section the block of tissue using the cryostat microtome. First, you need to mount your frozen tissue sample onto the piece called the chuck that fits into the microtome head and serves as the stage for your sample.
To start, place a small amount of embedding medium on the chuck and let it freeze. Place the chuck into the microtome head and shave off enough of the frozen medium to make a flat surface for your sample. Remove the chuck from the microtome head and place flat within the cryostat.
Pour embedding medium on the chuck and firmly. Place the brain block cut side down on the chuck slowly and completely. Embed the piece of brain in the mounting medium.
Place the chuck now mounted with the brain into the Microtome head section using the predetermined section sampling frequency. After making a section either mounted on a slide or add it to a specified series in the plate containing antigen preserve, be very careful to keep the sections in the order. You slice them so that later you can go back to the brain bank and take another systematic sample.
Once you have exhaustively sectioned the tissue and have placed the sections into wells, cover the plate with the lid, wrap the lid with param and place in a minus 20 degree Celsius freezer. Log the total number of sections in each series, the section sampling interval, and the number of series per animal. This log will be vital for keeping track of sections as you withdraw them from the brain bank for future experiments.
Systematically sampling in this manner has been a standard practice in our laboratory. For the past three years, we've had a great deal of success performing immunohistochemistry on material that has been stored in antigen preserve for three years without deterioration of the signal. Furthermore, as part of our brain bank, we have logged close to 20, 000 systematic sections of the non-human primate brain.
As part of our long-term research plans, We've just shown you how to systematically sample a biological specimen and preserve it as part of your long-term research plans. When doing this procedure, it is important to remember to keep a detailed log of the total number of series taken and the total number of sections per series. Also, if a section is lost or damaged, record that section number down in the logbook in order to maintain a systematic sampling procedure.
So that's it. Thanks for watching and good luck with your experiments.
