Immunohistochemistry

Hello Vielle, the methods given in the paper include details such as the specific antibodies used, how brains were prepared and sectioned etc.

Here is a detailed staining protocol:

IHC and Mounting

Day 1 (permeabilization and blocking)

1. In a 15mL falcon tube, make a master mix of permeabilization solution containing 3% BSA and 0.3% Triton X-100. Make enough for 0.5mL of solution in each well.
   1. Jordan usually stains 4 animals at a time which means 8 total wells of slices leading to a 4mL mastermix.
      1. Solution components for 4mL master mix (on post-it above Jordan’s bench)
         1. 3.6mL PBS
         2. 400uL 30% BSA (in “Antibodies & IHC box” on Jordan’s shelf in Mr. Freeze)
         3. 12uL Triton x-100 (in falcon tube on Jordan’s bench)
2. Remove PBS from wells using p1000 and leave slices behind.
3. Pipette permeabilization solution up and down in falcon tube to mix. 
4. Add 0.5mL of permeabilization solution to each well. Shake plate gently to make sure slices are not stuck together or to walls of well.
5. Cover plate in aluminum foil and place plate on shaker in 4C cold room overnight.

Day 2 (primary antibodies)

1. In a 15mL falcon tube, make a master mix of primary antibody solution containing 3% BSA, rabbit anti-VGLUT2 (1:500; Synaptic Systems, cat no. 135 403), chicken anti-GFP (1:1000; Aves Labs, cat no. GFP-1020), and 0.3% Triton X-100. Make enough for 0.5mL of solution in each well.
   1. Solution components for 4mL master mix (on post-it above Jordan’s bench)
      1. 3.6mL PBS
      2. 400uL 30% BSA 
      3. 4uL chicken anti-GFP (1:1000; stock in Sean’s antibody box in Erin’s fridge)
      4. 8uL rabbit anti-VGLUT2 (1:500; aliquots in “Antibodies & IHC” box on Jordan’s shelf in Mr. Freeze)
      5. 12uL Triton x-100
2. Remove permeabilization solution from wells using p1000 and leave slices behind.
3. Pipette primary antibody solution up and down in falcon tube to mix. 
4. Add 0.5mL of primary antibody solution to each well. Shake plate gently to make sure slices are not stuck together or to walls of well.
5. Cover plate in aluminum foil and place plate on shaker in 4C cold room overnight.

Day 3 (secondary antibodies)

1. Remove primary antibody solution from wells and leave slices behind.
   1. Sometimes, Jordan saves this if he has another batch of staining ready for primary in the next couple of days. Do not reuse more than once or twice.
      1. If reusing, place removed primary antibody solution in falcon tube in 4C cold room.
2. Wash slices by adding PBS to wells and placing on belly dancer at room temperature (covered) for 10 minutes. Do this 3 times.
3. Remove PBS from wells and leave slices behind.
4. In a 15mL falcon tube, make a master mix of primary antibody solution containing 3% BSA, goat anti-rabbit 647 (1:500; Invitrogen, cat no. A21245), goat anti-chicken 488 (1:500; Invitrogen, cat no. 11039), blue fluorescent Nissl stain 435 (1:250; Invitrogen, cat no. N21479), 10% goat serum, and 0.3% Triton X-100. Make enough for 0.5mL of solution in each well.
   1. Solution components for 4mL master mix (on post-it above Jordan’s bench)
      1. 3.2mL PBS
      2. 400uL 30% BSA 
      3. 400uL goat serum (goat serum box in Bieber chest freezer)
      4. 8uL goat anti-chicken 488 (common secondary antibodies box in hallway fridge)
      5. 8uL goat anti-rabbit 647 (common secondary antibodies box in hallway fridge)
      6. 16uL blue fluorescent Nissl stain (stock aliquots in “Vera primary antibodies” box in hallway -20C)
         1. Working aliquots in “Antibodies & IHC box in Mr. Freeze)
      7. 12 uL Triton x-100 
   2. Don’t forget that the secondaries are photosensitive. Keep them out of light as much as possible!
5. Pipette secondary antibody solution up and down in falcon tube to mix. 
6. Add 0.5mL of secondary antibody solution to each well. Shake plate gently to make sure slices are not stuck together or to walls of well.
7. Cover plate in aluminum foil and place plate on shaker in 4C cold room overnight.

Day 4 (mounting)

1. Remove secondary antibody solution from wells and leave slices behind.
2. Wash slices by adding PBS to wells and placing on belly dancer at room temperature (covered) for 10 minutes. Do this 3 times.
3. Remove the 3rd wash from the wells and add PBS again so slices can be taken out of wells for mounting.
4. Transfer slices from one well to a petri dish containing sink water to wash off PBS. Mount these slices onto a slide using sink water again.
   1. Mounting with sink water prevents the crystallization of salt on slide after drying.
5. Keep slide covered until dry. Jordan likes to place in a slide book.
6. Repeat with each of the other wells.
7. When a slices on slide are dry, use VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories, cat no. H-1400; Erin’s fridge) to mount coverslip onto slide. 
8. When done, place all slides in slide book and leave in 4C cold room for 15-20 min so mounting medium can harden. 
9. Store slides in a slide box on Jordan’s shelf in Mr. Freeze (-20C).