TITLE: Brain processing, slicing and immunohistochemistry protocol
AUTHORS: Andrew Hunter

[DESCRIPTION]
This is a step by step procedure from collecting brain samples to immunohistochemical staining and mounting brain slices. 

[STEPS]
SECTION: Perfusion fixation
1. While wearing proper PPE, set up surgical area in fume hood
SECTION: Perfusion fixation
2. Once a suitable period post-surgery has occurred (3+ weeks) the animal can be perfused 
SECTION: Perfusion fixation
3. Set up surgical area
SECTION: Perfusion fixation
4. Load ketamine-xylazine cocktail into injection syringe (0.2mL/mouse)
SECTION: Perfusion fixation
5. In physiological fume hood (separate from PFA-exposed hood) load isoflurane into holding chamber
SECTION: Perfusion fixation
6. Place mouse in isoflurane holding chamber until mouse breathing has slowed and running has stopped
SECTION: Perfusion fixation
7. Inject 0.2mL ketamine/xylazine IP and place animal back into original cage
SECTION: Perfusion fixation
8. Monitor breathing and foot-pinch twitch reflex
SECTION: Perfusion fixation
9. Once mouse reflex has stopped (should still be breathing) quickly move mouse to the surgical area
SECTION: Perfusion fixation
10. Restrain mouse using PrecisionGlide needles to hold down limbs
SECTION: Perfusion fixation
11. Cut mouse skin from to reveal fascia layer, do not yet make incision to reveal pleural space
SECTION: Perfusion fixation
12. Using fine surgical scissors, cut fascia to reveal organ space
SECTION: Perfusion fixation
13. Cut diaphragm and up sides of rib cage to reveal heart
SECTION: Perfusion fixation
14. 
Using resistance clippers, cut right atrium of the heart
SECTION: Perfusion fixation
15. Grip heart and insert cold PBS flat needle into left ventricle from the apex of the heart, begin to perfuse 10mL of PBS by hand at ~2mL/min
SECTION: Perfusion fixation
16. Begin flow of PFA at 120mL/hr, switch PBS needle for PFA needle using the same hole formed from the initial insertion
SECTION: Perfusion fixation
17. Set timer for 10min, when expires reduce flow to 100mL/hr for 15min
SECTION: Perfusion fixation
18. Set timer for 15 min, when expires reduce flow to 90mL/hr until 50mL is reached, 25min
SECTION: Perfusion fixation
19. Remove needle, turn mouse over (should be rigid due to PFA perfusion) and use thick scissors to sever head
SECTION: Perfusion fixation
20. Using fine surgical scissors, remove scalp to reveal skull
SECTION: Perfusion fixation
21. Use resistance clippers to gently cut skull without damaging brain, make incisions on skull at most rostral section of brain to enable skull to be peeled from brain
SECTION: Perfusion fixation
22. Use spatula to delicately remove brain and place into glass vial labelled with mouse ID, 4% PFA, initials and date
SECTION: Perfusion fixation
23. Refrigerate brain at 4°C for 24 hours
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
24. Set up work-station by gathering 6-well plate with PBS, a petri dish, and 6/12 glass vials (depending on if collecting both hemispheres)
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
25. Fill all of these with PBS, label the glass vials with the mouse ID, hemisphere genotype, initials, and date
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
26. Remove vibratome blade from manufacturer packaging and wash with ethanol followed by distilled water to remove protective oils

SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
27. Use hex-tool to tighten blade to the blade-holder so that it is straight and extends several millimeters beyond the black blade-guards
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
28. Do not attach blade to vibratome at this time
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
29. Plug in vibratome and turn on, check settings: Slice thickness is 70um, slice frequency is set at 9, slicing speed should be between “4” and “5”
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
30. Retrieve brain from 4°C Fridge, wash in PBS x 3 and dispose of waste in Liquid PFA waste disposal unit
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
31. Place brain into holding chamber and using a brush, gently orient the brain to be equally distributed along the sagittal axis
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
32. For sagittal slicing, take one Personna 0.012 HD Heavy Duty Single Edge Razor blade and insert into the sagittal guide slits, push down through brain while maintaining even force between sides of the blade, this will cut the brain into two halves
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
33. Take one hemisphere and place into a petri dish filled with PBS, put the other half back into glass vial of PBS and store at 4°C until ready to slice
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
34. Equip the scalpel with its blade and prepare to use
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
35. Using spatula, pick up the hemisphere by the bisected plane on the flat edge of the spatula
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
36. Using filter paper, dry the bisected plane, absorbing residual PBS
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
37. Paint a thin line of super glue onto the vibratome stage, then quickly use the flat side of the scalpel blade to push the onto the stage so that the bisected plane comes into full contact with the glue
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
38. Place stage in stage holder and fill with PBS
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
39. Attach vibratome blade to vibratome
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
40. Raise the stage so that the blade is several millimeters above the slice
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
41. Using V-Max, set front and back of continuous cutting using the limit-set button, should be set a few millimeters in front of and behind the most forward and back parts of the brain
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
42. Begin slicing, placing the first brain slice in well 1, and the following in well 2, going in 6 slice groups (slice 7 will be in well 1) to generate a 1/6 series of the brain per vial
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
43. Once complete, take slices from ONE WELL that should contain your ROI and observe for viral expression using the epifluorescent microscope (if applicable)
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
44. Return slices to their original well
SECTION: Brain Slicing - Vibratome Operation (24 hours post-perfusion)
45. Transfer slices
SECTION: Primary incubation
46. Place each series in a glass vial and rinse sections with PBS 3 times 
SECTION: Primary incubation
47. Add 1 mL of PBS-T with 2% Normal Donkey Serum (20 µL/mL) to each series and swirl briefly
SECTION: Primary incubation
48. Optional blocking step: leave slices in PBS-T and 2% Normal Donkey Serum at room temperature for 45–60 min
SECTION: Primary incubation
49. Add primary antibody to each series (see table 2)
SECTION: Primary incubation
50. Shake gently for 48–72 h at 4 ◦C (sections should barely revolve around the vial)

SECTION: Secondary incubation
51. Rinse sections with PBS 3 times before starting secondary reactions 
SECTION: Secondary incubation
52. Create necessary “Secondary Antibody Cocktail” consisting of 1 mL of PBS-T with 2% Normal Donkey Serum (20 µL/mL) and corresponding secondary antibody (refer to suggested antibody concentration). Volume of cocktail should be +1 to all reagents to avoid lack of volume due to pipette error (meaning 12 vials = 13mL PBS-T, 20uL NDS x 13, 4uL 2° x 13)
SECTION: Secondary incubation
53. Protect from light for all remaining steps. 
SECTION: Secondary incubation
54. Shake gently for 90 min at room temperature (sections should barely revolve around the vial) 
SECTION: Secondary incubation
55. Rinse sections with PBS 3 times before mounting 
SECTION: Secondary incubation
56. Using a glass petri dish, gently remove brain slices from 1 vial at a time and mount onto “name of glass slide” 
SECTION: Mounting slices and Labelling
57. Mount sections serially on slides with Prolong Diamond Anti-fade mounting media; protect slides from light and keep at 4 ◦C after 24 h drying at room temperature
SECTION: Mounting slices and Labelling
58. Proper slide labelling format is
 mouse ID – hemisphere
 series
 primary antibody
 secondary antibody
 genotype
 initials – date

SECTION: Mounting slices and Labelling
59. Insert new slide data and location into slide census spreadsheet