TITLE: Immunofluorescence and confocal imaging protocol
AUTHORS: taylor.panczyk

[DESCRIPTION]
In this protocol we detail the steps needed to obtain fixed brain tissue from mice to perform immunohistochemistry and image acquisition. In particular, we detail the perfusion, dissection, brain slicing, mounting and anti-ChAT immunolabeling. Here we also detail the steps for confocal images acquisition.

[STEPS]
SECTION: Dissection:
14. Decapitate the mouse with large surgical scissors.
SECTION: Tissue Fixation Through Perfusion:
1. Anesthetize the mice with a mixture of ketamine (50 mg/kg) and xylazine (4.5 mg/kg).
SECTION: Tissue Fixation Through Perfusion:
5. Make two additional skin incisions from the xiphoid process along the base of the ventral ribcage laterally.
SECTION: Tissue Fixation Through Perfusion:
13. Shift the pump tubing to PFA 4% and perfuse during 8 minutes.
SECTION: Tissue Fixation Through Perfusion:
12. Turn on the pump and perfuse 3 minutes with PBS-heparineat at 8 ml/min rate.
SECTION: Tissue Fixation Through Perfusion:
7. Grasp the cartilage of the xiphoid process with blunt forceps and raise it slightly to insert pointed scissors. Cut through the thoracic musculature and ribcage between the breastbone and medial rib insertion points and extend the incision rostrally to the level of the clavicles.
SECTION: Tissue Fixation Through Perfusion:
6. Gently reflect the two flaps of skin to expose thoracic field completely.
SECTION: Tissue Fixation Through Perfusion:
11. Cut the right atrium with scissors, and at the first sign of blood flow, begin the infusion of 1x slicing solution at 2-4 ml/min.
SECTION: Tissue Fixation Through Perfusion:
10. Secure the beating heart with fingers or blunt forceps, and immediately insert a blunt 27G syringe needle.
SECTION: Tissue Fixation Through Perfusion:
9. Clamp the reflected ribcage laterally with a hemostat to expose the heart.
SECTION: Tissue Fixation Through Perfusion:
8. Separate the diaphragm from the chest wall on both sides with scissor cuts.
SECTION: Dissection:
15. Cut down the midline to expose the skull.
SECTION: Dissection:
16. Make two lateral and one dorsal cut using sharp surgical scissors on the base of the skull.
SECTION: Dissection:
17. Cut the olfactory bulbs/optic nerve at rostral end of skull.
SECTION: Dissection:
18. Gently peel off the skull using blunt forceps.
SECTION: Dissection:
19. Once brain is fully exposed, remove it from the skull and place it in a vial containing PFA 4%.
SECTION: Dissection:
20. Leave the brain in PFA 4% for 4 hrs for post-fixation.
SECTION: Dissection:
21. Rinse the brain with PBS and transfer it to a vial containing PBS and store at 4 °C until slicing.

SECTION: Tissue Fixation Through Perfusion:
4. Make an incision through the abdominal skin.
SECTION: Tissue Fixation Through Perfusion:
2. Use toe pinch-response method to determine depth of anesthesia.
SECTION: Tissue Fixation Through Perfusion:
3. Place the animals on a styrofoam tray lying on the back with face upward.
SECTION: Brain Section:
22. Cut a 2 cm piece of the 20° agarose wedge and super glue it to the specimen plate of Leica VT1200S vibrotome.
SECTION: Brain Section:
23. Insert razor blade into blade holder.
SECTION: Brain Section:
24. Set parameters (speed, amplitude, and section thickness) on the Vibratome control panel.
SECTION: Brain Section:
25. Cut the brain in two halves along the midline.
SECTION: Brain Section:
26. Super glue the flat section (midline) onto the agarose wedge glued to the specimen plate so that the vibratome cuts along parasagittal plane with a 20° angle. Place the posterior part of the brain in the top of the agarose wedge. If bilateral sections are needed, super glue both hemispheres to the agarose wedge.
SECTION: Brain Section:
27. Play start to the vibrotome and cut the desired slices at 80 um thick.
SECTION: Brain Section:
28. With a pencil transfer the slices to PBS fillet 1.5 tubes.
SECTION: Brain Section:
29. Store the slices at 4 °C , in darkness, until mounting or immunolabeling. 

SECTION: Mounting:
30. Choose the desired section and carefully mount them in a slide with a pencil.
SECTION: Mounting:
31. Wait until PBS is almost completely dry. 
SECTION: Mounting:
32. Cover with mounting solution ProLong Diamond.
SECTION: Mounting:
33. Place a cover slip over the mounting solution and carefully distribute it below the cover slip, trying to avoid bubbles over the brain sections. 
SECTION: Mounting:
34. Maintain all the slides in darkness. 
SECTION: Immunolabeling alpha-ChAT: Primary ab
35. Wash at RT 3-5 x 5 minutes in PBS
SECTION: Immunolabeling alpha-ChAT: Primary ab
36. 1 hr blocking at RT on shaker in 1x PBS + 5% Normal horse serum + 0.3% Tx-100.
SECTION: Immunolabeling alpha-ChAT: Primary ab
37. Overnight at 4 degrees in the blocking solution + primary (1:1000) (goat anti-chat - millipore AB144).
SECTION: Immunolabeling alpha-ChAT: Primary ab
38. Wash at RT 3-5 x5 minutes in PBS.
SECTION: Immunolabeling alpha-ChAT: Secondary ab
39. Secondary in PBS (1:500) at RT for 2-4 hours (closer to 2, 4 is pushing it) (Alexa Green or Red anti-goat).
SECTION: Immunolabeling alpha-ChAT: Secondary ab
40. Wash at RT 3-5 x 5 minutes in PBS.
SECTION: Immunolabeling alpha-ChAT: Secondary ab
41. Mounting.
SECTION: Confocal Imaging:
42. Place the slide in the slide holder of the Olympus FV10i confocal laser scanning microscope.
SECTION: Confocal Imaging:
43. Turn on the power of laser, miscroscope, and computer. 
SECTION: Confocal Imaging:
44. Open the software.
SECTION: Confocal Imaging:
45. Set lightpath to locate brain slice under 10x lens.
SECTION: Confocal Imaging:
46. Once the brain slice is located, switch the lightpath to camera.
SECTION: Confocal Imaging:
47. Check for lasers of interest and make sure they are in use. 
SECTION: Confocal Imaging:
48. Switch to 40x or 60x lens for higher magnification images. 
SECTION: Confocal Imaging:
49. Save images. 
SECTION: Confocal Imaging:
50. Use FIJI (NIH) to adjust images for brightness, contrast, and pseudo-coloring.