Mar 20, 2023
Synaptic immunohistochemistry - wholemount via acetone permeabilization
- Anya Suppermpool1,
- FishFloorUCL1
- 1University College London
Protocol Citation: Anya Suppermpool, FishFloorUCL 2023. Synaptic immunohistochemistry - wholemount via acetone permeabilization. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge38q7l47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: March 08, 2023
Last Modified: March 22, 2023
Protocol Integer ID: 78330
Abstract
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:
Materials
PO4buffer
8 parts0.1MNaH2PO4
2 parts0.1MNa2HPO4
BT buffer (make fresh)
1.0gsucrose
18.75µL0.2MCaCl2
PO4buffer to15mL
BT fix (make fresh)
1.2mLPO4buffer
4.8mLBT buffer
2mL16% PFA (used bought Thermofisher fix)
PBS/BSA/DMSO
50mLPBS
0.5gBSA
500µLDMSO
Reagents
Reagents
PO4buffer
8 parts0.1MNaH2PO4
2 parts0.1MNa2HPO4
BT buffer (make fresh)
1.0gsucrose
18.75µL0.2MCaCl2
PO4buffer to15mL
BT fix (make fresh)
1.2mLPO4buffer
4.8mLBT buffer
2mL16% PFA (used bought Thermofisher fix)
PBS/BSA/DMSO
50mLPBS
0.5gBSA
500µLDMSO
Day 1
Day 1
Tricaine, Dechorionated beforehand (2dpf)
Fix 1.5-2h in BT fix (*use bought fix) at 4C
(less fix time higher SNR but mushy) 5h fixation works for 5dpf – 9dpf larvae, time may need to be adjusted for different ages
Replace fixative with PO4 buffer.
(Optional) Store at 4C overnight (shaking not necessary)
Wash for 5’ RT with shaking if continuing with permeabilization and blocking in same day
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
Transfer larvae to glass vials using glass Pasteur pipette.
Larvae will be sticky so be careful while transferring between tubes!
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
Wash with cold acetone 5’ at -20C (put in freezer)
Wash with dH2O 5’ RT with rocking.
This step can go slightly longer (~10’) if necessary
Wash with PO4, 5’ at RT (Lavinia’s original protocol extra step)
Block >2h RT in PBS/BSA/DMSO + 2% goat serum.
(optional) can go overnight at 4C
Incubate in primary antibodies in PBS/BSA/DMSO 4C overnight.
Use ~1mL antibody mix per tube
Day 2
Day 2
Remove primary antibody mix.
Add 1:1000 20%NaN3to mix if saving for future use
Wash 5X+ withPBS/BSA/DMSO >20’ RT with rocking.
(optional) any washes after antibody incubation can go overnight at 4C if necessary
Incubate with secondary antibodies inPBS/BSA/DMSO 2-3h at RT or overnight at 4C in the dark
Day 3
Day 3
Wash 3X withPBS/BSA/DMSO >20’ RT with rocking
(Wash withPBS/BSA/DMSO + DAPI (1:2000 dilution) >20’ RT with rocking)
Wash withPBS>20’ RT with rocking like 5x
Move to glycerol – 20%,40%,60%,80%
Mount and image using glycerol lens (did 20x before)
| Reagents | B | C | |
| Anti-MAGUK | 1:500 | MABN72 Anti-pan-MAGUK, clone K28/86 | |
| Fix | #28906 | ThermoFisher (from Ana Faro) | |
| tRFP | 1:500 | Anti-tRFP Rabbit Polyclonal (AB233 - evrogen) |