Immunofluorescence for Primary Brain Cell Cultures
Ashley V Kumar, Francesca Telese
Abstract
Here we describe a protocol to detect specific proteins by immunofluorescence on cells cultured as monolayers. The protocol has been tested on primary cortical neurons and primary astrocytes isolated from mouse cortical tissues.
Before start
Steps
Day 1: Fixation, Permeabilization, Blocking, Primary Antibody
Fixation. Aspirate culture media and wash once with ice-cold 1X PBS. Add 4% Formaldehyde fixation buffer. Incubate at room temperature (RT) for 15 min. Use an orbital shaker using low speed.
Wash 3 times with ice-cold PBS. Incubate for 5 min on the orbital shaker at RT
Wash 1 ロ
Wash 2 ロ
Wash 3 ロ
Permeabilization: Add 500uL of 0.3% Triton in PBS per well (for 8-wells, reduce to 250uL), incubate at RT 5 min on the orbital shaker
3X Washes: Ice-cold PBS for 5 min (Incubate at RT, Orbital shaker)
Wash 1 ロ
Wash 2 ロ
Wash 3 ロ
Blocking: Add 600uL of 100% Superblocking Buffer. Incubate at RT for 1h with orbital shaker
Primary antibody hybridization: Dilute antibody in antibody hybridization buffer. Incubate o/n at 4℃ or 1 hour at RT using an orbital shaker
Suggested volumes: 600μL/well for 4-wells & 300μL/well for 8-wells
Day 2: Secondary antibody
Remove Primary Ab hybridization buffer. Antibodies can be stored at 4C and re-used.
3X Washes: Ice-cold PBS for 5 min (Incubate at RT, Orbital shaker)
Wash 1 ロ
Wash 2 ロ
Wash 3 ロ
Secondary antibody hybridization: Dilute secondary antibody in antibody hybridization buffer. Incubate at RT for 1 hr using an orbital shaker.
Suggested volumes: 500μL per well for a 4-well slide
Secondary antibodies are coupled with fluorophores. Perform these steps in the dark using dark chambers
3X Washes: Ice-cold PBS for 5 min (Incubate at RT, Orbital shaker)
Wash 1 ロ
Wash 2 ロ
Wash 3 ロ
Stain every well with Hoechst 33342 diluted 1:1000 in PBS. Incubate at RT for 3 min on an orbital shaker
3X Washes: Ice-cold PBS for 5 min (Incubate at RT, Orbital shaker)
Wash 1 ロ
Wash 2 ロ
Wash 3 ロ
Day 2: Slides Mounting
Remove silicone gaskets from the glass slides. With a Kimwipe, remove the excess of PBS without touching the surface with the cells
Spread ~200uL of Prolong Glass antifade mounting medium across the whole slide. Slowly cover the glass slide with a glass coverslip and avoid making air bubbles. Seal the coverslip to the slide with clear nail polish
Let the slides dry for 24 hr at RT in the dark before the fluorescent microscopy analysis image. For long-term storage, keep the slides at 4℃ in dark chambers.
