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