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Archive for June, 2009

Abstract

June 23, 2009 1 comment
Categories: Uncategorized

June 22nd, 2009

June 22, 2009 14 comments

Today I finished taking pictures through the microscope, which came to a grand total of 205 images. With this many pictures I will be able to gather tons of useful data. I havent really explained why we are interested in the AT1 receptors yet on this blog so I’ll do a brief overview now. The animals (rats and hampsters) have been split into four groups (not mixed between species). The groups consist of the hypertensive (high blood pressure) animals, the normotensive (regular blood pressure), the captopril-treated hypertensive group (high blood pressure given a drug to lower it), and the hypertensive group that was briefly given captopril then taken off of it. The reason why we are interested in this study is because when you give the hypertensive rats captopril to lower their blood pressure but then stop giving the drug to them you would expect that their blood pressure would go back to a hypertensive range (about 160 mmHg). Instead it stays about 20-30 mmHg lower than it was originally, even though the drug has not been administered for weeks. We are trying to find a change in the amount of these AT1 receptors (proteins) in the brain to see if we can explain the lowered blood pressure off the drug. The simplified version behind raising your blood pressure is when your body binds angiotensin II (a neurotransmitter) to its receptors, AT1, and this causes vasoconstriction (narrowing of the blood vessels) to increase the pressure. Its kind of alot of information to try to decypher at one time but thats a brief overview of the project I’m working on right now. Tomorrow I am going to start gathering the data which I do have some pictures to explain it visually.

 

Scale 10x copy

Here’s a picture of the scale at 10x magnification. This enables me to measure the area of the brain regions by setting a scale from pixels to mm.

 

 

 

Area Measurements

This is a picture of the Hypoglossal Nucleus at 10x. I am measuring the area inside the yellow dotted lines.

 

 

 

Quadrants 20x

This is a picture of the ventral and dorsal regions that I am counting neurons in. This is of the hypoglossal nucleus at 40x. Some of the neurons are circled in yellow.

Categories: AT1 Study

Brainstem Regions and Pictures

June 17, 2009 2 comments

After I completed day 2 for the mounted amplification technique on  the hampster slices I took pictures through the microscope of the regions we are studying.

 

Here’s a picture at 10x magnification on the brainstem where you can see the different brain regions conveniently on one slice. The darker (brown) regions are marked, which means that they have AT1 receptors within them.

Blog Pic of 5-3 B4 10x

 

 

 

 

Here’s the above picture at 40x magnification in the hypoglossal nucleus.  You can see that the neurons have alot of AT1’s on them by the dark brown color. 

Blog Pic of Motor Neurons at 40x

Categories: AT1 Study

Visualizing AT1 Receptors Using Immunohistochemistry

Here’s a condensed version of the modified protocol we assembled to visualize the AT1 receptors on the brain tissues of hampsters….

  1. Wash the slides 3x in 0.1M PBS for 5 min
  2. Wash in .075% H2O2 for 30 min in .1M PBS
  3. Wash the slides 3x in 0.1M PBS for 5 min
  4. Incubate in the Primary Antibody (1:500) in a buffer solution containing 10% goat serum, 0.1% triton X-100, 0.05% tween-20 in 0.1M PBS for 2 hours at room temp then for another 48 hours at 4°C.
  5. Wash the slides 3x in 0.1M PBS for 5 min
  6. Incubate in the Secondary Antibody (1:250) for 50 min at room temp in the buffer solution
  7. Wash the slides 3x in 0.1M PBS for 5 min
  8. Incubate in Streptavidin-Horseradish Peroxidase (1:150) in a TNB blocking buffer solution for 30 min.
  9. Wash 3x in TNT wash for 5 min
  10. Amplification for 5 min in DMSO (1:50) in amplification diluent.
  11. Wash the slides 3x in 0.1M PBS for 5 min
  12. Incubate in ABC kit for 30 min
  13. Wash the slides 3x in 0.1M PBS for 5 min
  14. Incubate in DAB (6000µl 0.1M PBS, 2.4mg DAB, 15µl) for 2-10 min until desired coloration is obtained