Synaptic immunohistochemistry - wholemount via acetone permeabilization

PO4buffer ₄buffer

8 parts0.1MNa₂2P₄4

2 parts0.1M₂a2H₄O4

BT buffer (make fresh)

1.0gsucrose

18.75µL0.2MCa₂l2 ₄PO4buffer to15mL

BT fix (make fresh)

1.2mLP₄4buffer

4.8mLBT buffer

2mL16% PFA (used bought Thermofisher fix)

PBS/BSA/DMSO

50mLPBS

0.5gBSA

500µLDMSO

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

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

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)

A	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)