[Protocol] Immunostaining protocol from Tony (Southall)

Published on Author Anne Petzold

Immunolabelling of larval and adult brains

  • Dissect brains in PBS
  • Fix with 4% formaldehyde in PBS containing 0.5 EGTA, 5mM MgCl2 (see below) @RT for 25 min.
  • Wash 3 x 5 min. (approx.) in PBST (0.3% Triton-X100 in PBS).

BREAK Can store in 4% PBT @RT or 4 DEG.

  • 4) Preincubate in 90% PBST / 10% Normal Goat Serum (NGS) for >= 15 min.
  • 5) Incubate in 1st antibody diluted in PBT/NGS O/N @4oC.
  • 6) Wash 3 x 5 min. (approx.).
  • 7) Incubate in 2nd antibody diluted in PBST for 2-3 hrs @RT.
  • 8) Wash 2 x 5min in PBST.
  • 9) Wash 2 x 10 min in PBST.
  • 10) Mount in Vectashield.

Fix solution


  • 250ml PBS
  • 0.25ml 0.5M EGTA* pH8.0
  • 1.25ml 1M MgCl2

Stock formaldehyde is at 40%

EGTA 0.5M stock solution (from OpenWetWare)

100 ml solution

  • 19g EGTA (MW 380g/mol)
  • ddH2O to 90ml
  • adjust pH 7.5/8.0 with solid NaOH (>4g)
  • adjust volume to 100ml

Note: EGTA will not go into solution without NaOH. Once the pH has been raised sufficiently it dissolves quickly. For pH 7.5 the exact amount required is slightly above 4g. Add 3.5-4g immediately, then proceed carefully not to overshoot the desired pH.

MgCl2 1M

100 ml solution

  • 20.33 g MgCl2.6H2O (Molecular weight = 203.30)
  • ddH2Oto 80 ml
  • adjust volume to 100 ml once dissolved

4% PFA

100 ml solution

  • 100 ml PBS
  • 4 g PFA
  • TOXIC, cover with parafilm, shake, put on hotplate to warm up until solution is clear
  • when cooled down, keep at 4 DEG

*Ethylene glycol tetraacetic acid (EGTA) is a common buffer ingredient due to its chelating activity. It is similar to the better known EDTA, but has a much higher affinity for calcium ions than for magnesium ions. Buffers made with EGTA are used in some cases to mimic the environment inside living cells where calcium ions are usually at least a thousandfold less concentrated than magnesium ions. (wikipedia)