TITLE: Brain slicing
AUTHORS: rabdelha  

[DESCRIPTION]
This protocol details brain slicing.

[GUIDELINES]

Vibratome Protocol Notes

Sectioning notes from the literature

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35723
https://www.cell.com/molecular-therapy-family/nucleic-acids/pdfExtended/S2162-2531(17)30241-X

Look striatum bregma 0.35mM



[STEPS]
SECTION: Embedding the brain:
1. Perform a gross coronal cut using the 1 mm matrix at a site either anterior or posterior of the region of interest, ~2mm distal from the region of interest. 
SECTION: Embedding the brain:
4. Dry the flat part of the brain repeatedly on a kimwipe. 
SECTION: Embedding the brain:
5. Place in the plastic square mold, with the now flat portion of the brain lying face-down, and press against the mold to eliminate any bubbles.
SECTION: Embedding the brain:
6. Using the plastic dropper, put the agarose/azide solution around the brain until completely submerged. 
SECTION: Embedding the brain:
7. Once submerged, place on ice or place in the fridge until completely solid. 
 

SECTION: Embedding the brain:
8. Once solid, pop out of the molds and dry off.
SECTION: Embedding the brain:
9. Take a sharp razor and slice the excess agar off to make a small cube. 
 

SECTION: Embedding the brain:
10. On the circular stage, paint a generous layer of superglue generously in the middle. 
SECTION: Embedding the brain:
11. Place the brain in the pool of superglue, face down, and swirl around to eliminate any bubbles and make full contact.
SECTION: Embedding the brain:
12. Let dry. 
 

SECTION: Mounting the Stage
13. 
Lock the rectangular stage pool in the place by moving the clamp straight up from its left-pointing position.   

SECTION: Mounting the Stage
14. Screw the razor blade into the clamp.
SECTION: Mounting the Stage
15. Load the blade in first and then screw it tightly in. 
SECTION: Mounting the Stage
16. Use the screw to attach the blade holder into the vibratome.
SECTION: Mounting the Stage
17. Screw the circular stage on and orient it such that the cube of agarose is parallel to the razor blade.
 

SECTION: Mounting the Stage
18. Submerge everything in PBS (no azide) to cover the brain cube.
SECTION: Mounting the Stage
19. The knob to the top left increases the speed of slicing. The vibration frequency should be between 8 and 10.


  
-/+ control the thickness (z) direction of slices.
∑ is the cumulative microns since the start of the slicing.
Single – one slice
Continuous – continuous slicing
SECTION: Slicing
20. Orient the stage using the upper switch such that the razor blade’s path just barely grazes the top of the agarose cube. 
 

SECTION: Embedding the brain:
3. Place in the plastic square mold, with the now flat portion of the brain lying face-down, and press against the mold to eliminate any bubbles.
SECTION: Embedding the brain:
2. Dry the flat part of the brain repeatedly on a kimwipe. 
SECTION: Slicing
21. Definitely aim start cutting >1mm anterior/posterior of your region of interest (depending on the direction your slices are starting from), and aim to finish >1 mm beyond your region of interest. 
 

SECTION: Slicing
22. Use the button containing an up arrow and a down arrow to set the upper and lower limits of the razor path. 
SECTION: Slicing
23. The first press of the button sets the upper limit (above the agarose) and the second press of the button sets the lower limit (below the agarose). 
SECTION: Slicing
24. Using the individual arrow keys, set the thickness of the slice (in microns) and hit start. 
 

SECTION: Slicing
25. Pause can be used, provided a slice is not in progress.
SECTION: Slicing
26. To change the slice thickness, hit stop, then change the thickness, then hit start. 
 

SECTION: Cleanup
27. Hit stop.
SECTION: Cleanup
28. Manually change the stage height and push the razor back.

SECTION: Cleanup
29. Unscrew the blade holder and remove the blade (these are disposable).
SECTION: Cleanup
30. Remove the circular stage, and use a razor blade to shave off residual agarose and glue.
SECTION: Cleanup
31. Unlock the rectangular stage and pour out the PBS into the catch container.
SECTION: Cleanup
32. If not slicing another brain, place the circular stage, screw for the blade holder, and the blade holder in the rectangular pool and fill with MQ H2O. 
SECTION: Cleanup
33. While the parts are soaking, spray a paper towel with ethanol and wipe down the vibratome. 
SECTION: Cleanup
34. Pour out the water from the bath.
SECTION: Cleanup
35. Cleanup all mess, turn off the vibratome, and replace the cover.
SECTION: Sectioning Notes
36. Going to section the following brain regions


SECTION: Sectioning Notes
37. Want to have 5-8 brains per experimental group.
SECTION: Sectioning Notes
36.1. CB – no activation
SECTION: Sectioning Notes
36.4. Frontal cortex- no activation
SECTION: Sectioning Notes
36.3. STR – activation
SECTION: Sectioning Notes
36.2. SN- activation; focus on the SnPr
SECTION: Sectioning Notes
38. Antibodies to use:

TH
IBA1
A-synuclein (Ps129)
DAPI
SECTION: Sectioning Notes
39. Making gross cut of brain:

 
SECTION: Sectioning Notes
39.1. Plan is to cut at the anterior portion of the hypothalamus; one half will contain the striatum and frontal cortex and the other will contain the SN and the cerebellum.
SECTION: Sectioning Notes
39.3. Mount both halves of the brain and then slice them together.
SECTION: Sectioning Notes
39.2. From there we will do coronal slicing at   slices.

SECTION: Sectioning Notes
40. Splitting brain:
SECTION: Sectioning Notes
40.1. A good approach would be to split the brain into 4 wells.
 
SECTION: Sectioning Notes
40.4. For an experiment you pick one well and do the staining on all the slices of that one well; that way you can go back and re-stain the other 3 wells at a future time point if necessarily. 
SECTION: Sectioning Notes
40.5. Next you need to mount the slices and they must be mounted in the correct order that they appear in the brain.
SECTION: Sectioning Notes
40.2. First slice in well 1, second slice in well 2, etc. 
SECTION: Sectioning Notes
40.3. This way each well will represent a whole brain (more or less) with the space between slices being 200uM.
SECTION: Sectioning Notes
41. # of slices to look at per region:
SECTION: Sectioning Notes
41.1. Its really important to be consistent with the regions you are looking at and taking data from; need to be consistent among images and between brains.
 
SECTION: Sectioning Notes
41.2. The best thing to do is find 2-4areas for each region of the brain that are in sequential order and take several pictures of each area. 
SECTION: Sectioning Notes
41.4. Next, take a high mag pic and then quantify microglial activation, a-syn aggregation, etc. from that high mag picture. 
SECTION: Sectioning Notes
41.3. For taking pictures, you should first take a low mag pic of the area to look for any obvious global changes between different experimental groups.
SECTION: Sectioning Notes
42. Data collection:
SECTION: Sectioning Notes
42.1. Take a zoomed in picture of the 2-4 different slices per brain area and you can then combine data from those 2—4 different sections.

SECTION: Sectioning Notes
42.2. Can combine data from several sections that are several bregma points apart.