TITLE: Synaptic immunohistochemistry - wholemount via acetone permeabilization
AUTHORS: Anya Suppermpool, FishFloorUCL

[DESCRIPTION]
Whole-mount Immunohistochemistry – acetone permeabilization 

Works with anti-MAGUK ab.

Modified from M Westerfield protocol. 
Lavinia Sheets: T. Nicolson Lab September, 2010 
Revised August 2011 
Anya Suppermpool Synaptic (MAGUK) Version 2018

Anya's Benchling Protocol Archive at:
https://benchling.com/s/prt-nLJhuyARGTPcTi887wL1?m=slm-FGIOfoYTo8bWHpVnUZ0v

[STEPS]
SECTION: Day 1
5. Tricaine, Dechorionated beforehand (2dpf) 

SECTION: Day 1
6. Fix 1.5-2h in BT fix (*use bought fix) at 4C

SECTION: Day 1
7. Replace fixative with PO4 buffer.
(Optional) Store at 4C overnight (shaking not necessary)
SECTION: Day 1
6.1.  (less fix time higher SNR but mushy) 5h fixation works for 5dpf – 9dpf larvae, time may need to be adjusted for different ages
SECTION: Day 1
8. Wash for 5’ RT with shaking if continuing with permeabilization and blocking in same day
SECTION: Day 1
9. Chill a small volume (~50mL glass bottle) acetone at -20C at least 20’ prior to perm steps.
(optional) if rushed/forgotten, put acetone in -80C for a few minutes, but this could lead to over-permeabilization
SECTION: Day 1
10. Transfer larvae to glass vials using glass Pasteur pipette.
 Larvae will be sticky so be careful while transferring between tubes!
SECTION: Day 1
11. Wash with dH2O 5’ RT with rocking.
*Timing is critical for permeabilization steps*—process tubes in same order for each step and adjust volume of solutions to allow for quick handling 
SECTION: Day 1
12. Wash with cold acetone 5’ at -20C (put in freezer)
SECTION: Day 1
13. Wash with dH2O 5’ RT with rocking.
This step can go slightly longer (~10’) if necessary
SECTION: Day 1
14.  Wash with PO4, 5’ at RT (Lavinia’s original protocol extra step)
SECTION: Day 1
15. Block >2h RT in PBS/BSA/DMSO + 2% goat serum.
(optional) can go overnight at 4C
SECTION: Day 1
16.  Incubate in primary antibodies in PBS/BSA/DMSO 4C overnight.
Use ~1mL antibody mix per tube
SECTION: Day 2
17. Remove primary antibody mix.
Add 1:1000 20%NaN3to mix if saving for future use
SECTION: Day 2
18. Wash 5X+ withPBS/BSA/DMSO >20’ RT with rocking.
(optional) any washes after antibody incubation can go overnight at 4C if necessary

SECTION: Day 2
19. Incubate with secondary antibodies inPBS/BSA/DMSO 2-3h at RT or overnight at 4C in the dark
SECTION: Day 3
20. Wash 3X withPBS/BSA/DMSO >20’ RT with rocking
(Wash withPBS/BSA/DMSO + DAPI (1:2000 dilution) >20’ RT with rocking)
SECTION: Day 3
21. Wash withPBS>20’ RT with rocking like 5x
SECTION: Day 3
22. Move to glycerol – 20%,40%,60%,80%
SECTION: Day 3
23. Mount and image using glycerol lens (did 20x before)
SECTION: Day 3
24.  
Anti-MAGUK1:500MABN72 Anti-pan-MAGUK, clone K28/86tRFP1:500Anti-tRFP Rabbit Polyclonal (AB233 - evrogen)Fix#28906ThermoFisher  (from Ana Faro)
 
SECTION: Reagents
1. PO4buffer
8 parts0.1MNaH2PO4
2 parts0.1MNa2HPO4
SECTION: Reagents
2. BT buffer (make fresh)
1.0gsucrose
18.75µL0.2MCaCl2
PO4buffer to15mL
SECTION: Reagents
3. BT fix (make fresh)
1.2mLPO4buffer
4.8mLBT buffer
2mL16% PFA (used bought Thermofisher fix)
SECTION: Reagents
4. PBS/BSA/DMSO
50mLPBS
0.5gBSA
500µLDMSO