GFP Amplification Immuno

This protocol uses a combination of a conjugated GFP-primary antibody coupled with an Alexa Fluor 488 secondary to achieve robust amplification of endogenous GFP in mouse brain sections. Was optimized for tissue fixed in 4% PFA overnight, followed by cryoprotection in 30% sucrose / PBS solution prior to sectioning at 40-50 um on cryostat.

Reagents

Primary antibody: Rabbit Anti-GFP, AlexaFluor 488 conjugate (Fisher A21311)
Secondary antibody: Goat Anti-Rabbit AlexaFluor 488 (ThermoFisher, A11008)
Blocking solution: 3% normal goat serum, 0.3% Triton X-100, in PBS. For serum, the less expensive non-sterile stuff is fine

Protocol

Note that all steps assume gentle agitation on shaker

Day 1

• Incubate in blocking solution for 1 hour at room temp.
• Incubate overnight in primary antibody (1:1000) diluted in blocking solution at room temp.

Day 2

• Rinse 3x in PBS, 10 min each
• Incubate for 2 hours in secondary antibody (1:500) diluted in blocking solution at room temp.
• Rinse 3x in PBS, 10 min each
• Mount, coverslip, and enjoy