Immunofluorescence staining, vibratome sections
Veerle Baekelandt, María Sanchiz Calvo, Eduard Bentea
Abstract
Protocol for performing immunofluorescence staining on free-floating vibratome cut brain sections from rats or mice.
Steps
Day 1
Transfer sections to be stained to a new 24-well plate filled with 1X PBS. All incubations are with 500µL solution per well unless stated otherwise.
1X PBS rinse.
Antigen retrieval step (using oven).
Incubate sections in antigen retrieval solution (10 mM citrate buffer pH 6.0 ; see recipe in Materials) at 80oC for 0h 30m 0s.
Place the well plate in the same buffer on ice for 0h 20m 0s.
1X PBS rinse.
Wash sections in 1X PBS for 0h 5m 0s at room temperature on wobbler.
Wash sections in 1X PBS for 0h 5m 0s at room temperature on wobbler.
Incubate sections with 250µL blocking solution (PBS-T + 10% donkey serum) per well for 1h 0m 0s at room temperature on wobbler. (PBS-T = PBS + 0.1% Tergitol)
Dilute primary antibodies at the required concentration in PBS-T + 10% donkey serum.
Incubate sections with 250µL primary antibodies overnight at room temperature.
Day 2
1X PBS-T rinse.
Wash sections in 1X PBS-T for 0h 5m 0s at room temperature on wobbler.
Wash sections in 1X PBS-T for 0h 5m 0s at room temperature on wobbler.
Dilute secondary antibodies at the required concentration in PBS-T.
Incubate sections with 250µL secondary antibodies for 2h 0m 0s at room temperature (in the dark).
1X PBS rinse.
Wash sections in 1X PBS for 0h 5m 0s at room temperature on wobbler.
Wash sections in 1X PBS for 0h 5m 0s at room temperature on wobbler.
Briefly rinse sections in 1/2 PBS + 1/2 AD and allow to dry.
Mount with Mowiol.
