Answer:
Day 1. 
1) Wash sections 4X with gentle rocking/agitation at 4ºC, 5 min each in ice-cold 0.1 M PB (0.1M Na 
Phosphate pH 7.4). 
2) Block sections in vehicle (10% normal goat serum in 0.1 M PB+ 0.3% Triton X-100) for 1 h with 
gentle rocking/agitation at 4ºC 
3) Incubate sections in NeuroMab diluted in ice-cold vehicle. Dilution factors for NeuroMabs should 
be determined empirically, but generally NeuroMab tissue culture supernatants should be used at 
1:2-1:10, purified NeuroMab IgG from 1-20 µg/ml. Incubate sections overnight at 4ºC with gentle 
rocking/agitation. 
Day 2. 
4) Wash sections 4X with gentle rocking/agitation at 4ºC, 5 min each in ice-cold vehicle. 
5) Incubate sections in appropriate fluorescent goat anti-mouse secondary (e.g., Alexa Fluor 488 
goat anti-mouse IgG Invitrogen A11001 at 1:2000) for 1 h with gentle rocking/agitation at 4ºC. 
From this step on the sections should be protected from room light. 
6) Wash sections with gentle rocking/agitation at 4ºC, 5 min in vehicle. 
7) Wash sections with gentle rocking/agitation at 4ºC, 5 min in 0.1 M PB. 
8) Wash sections with gentle rocking/agitation at 4ºC, 5 min in 0.05 M PB. 
9) Mount on gelatin-coated microscope slides (floating in 0.05 M PB in a 15 ml Petri dish). 
10) Air dry 
11) Put 15 µl mounting media (e.g., Prolong Gold antifade reagent, Invitrogen P36930) on each 
section and coverslip. 
12) Seal with nail polish. 
 
from the NeuroMab website.