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AS_MAGUK synaptic antibody IHC Wholemount
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AS_MAGUK synaptic antibody IHC Wholemount

Introduction

Whole-mount Immunohistochemistry – acetone permeabilization Modified from M Westerfield protocol. Lavinia Sheets: T. Nicolson Lab September, 2010 Revised August 2011 Anya Suppermpool Synaptic (MAGUK) Version 2018

Materials

  • Solutions:
    • PO4 buffer
      • 8 parts 0.1M NaH2PO4
      • 2 parts 0.1M Na2HPO4
    • BT buffer (make fresh)
      • 1.0g sucrose
      • 18.75ul 0.2M CaCl2
      • PO4 buffer to 15 ml
    • BT fix (make fresh)
      • 1.2ml PO4 buffer
      • 4.8ml BT buffer
      • 2ml 16% PFA (used bought Thermofisher fix)
    • PBS/BSA/DMSO
      • 50ml PBS
      • 0.5g BSA
      • 500ul DMSO

    Procedure

    • Day 1
    1. Tricaine, Dechorionated beforehand (2dpf)
    1. 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
    1. Replace fixative with PO4 buffer
    • (Optional) Store at 4C overnight (shaking not necessary)
    1. Wash for 5’ RT with shaking if continuing with permeabilization and blocking in same day
    1. 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
    1. Transfer larvae to glass vials using glass Pasteur pipette
    • Larvae will be sticky so be careful while transferring between tubes!
    1. 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
    1. Wash with cold acetone 5’ at -20C (put in freezer)
    1. Wash with dH2O 5’ RT with rocking
    • This step can go slightly longer (~10’) if necessary
    1. Wash with PO4, 5’ at RT (Lavinia’s original protocol extra step)
    1. Block >2h RT in PBS/BSA/DMSO + 2% goat serum
    • (optional) can go overnight at 4C
    1. Incubate in primary antibodies in PBS/BSA/DMSO 4C overnight
    • Use ~1ml antibody mix per tube
    • Day 2
    1. Remove primary antibody mix
    • Add 1:1000 20% NaN3 to mix if saving for future use
    1. Wash 5X+ with PBS/BSA/DMSO >20’ RT with rocking
    • (optional) any washes after antibody incubation can go overnight at 4C if necessary
    1. Incubate with secondary antibodies in PBS/BSA/DMSO 2-3h at RT or overnight at 4C in the dark
    • Day 3
    1. Wash 3X with PBS/BSA/DMSO >20’ RT with rocking
    • (Wash with PBS/BSA/DMSO + DAPI (1:2000 dilution) >20’ RT with rocking)
    1. Wash with PBS >20’ RT with rocking like 5x
    1. Move to glycerol – 20%,40%,60%,80%
    1. Mount and image using glycerol lens (did 20x before)
    • Antibodies and fix
    Table1
    Collapse table
    A
    B
    C
    1
    		Anti-MAGUK1:500MABN72 Anti-pan-MAGUK, clone K28/86
    2
    		tRFP1:500Anti-tRFP Rabbit Polyclonal (AB233 - evrogen)
    3
    		Fix#28906ThermoFisher (from Ana Faro)
    3 rows

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