TITLE: Immunofluorescence for Primary Brain Cell Cultures
AUTHORS: Ashley V Kumar, Francesca Telese

[DESCRIPTION]
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.

[GUIDELINES]
This protocol has been tested for primary astrocytes and cortical neurons cultured in 4- or 8-well chambers slides. 

For astrocytes, coat slides with Poly-D, L-lysine hydrobromide (PDLL) (Sigma #P9011)  for 1 hour at 37C and then wash twice with H2O.

For cortical neurons, coat with Poly-l-ornithine: Cat. #: P3655, Sigma and Laminin: Cat. #: L2020, Sigma for 2 hours at 37C. Perform 2 quick rinses with 1X PBS. Do not let the slides dry. Keep the slides well submerged in 1X PBS at RT until plating. Aspirate PBS and perform 2 quick rinses with ddH2O right before plating.




[STEPS]
SECTION: Day 1: Fixation, Permeabilization, Blocking, Primary Antibody
1. 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. 
SECTION: Day 1: Fixation, Permeabilization, Blocking, Primary Antibody
2. 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

SECTION: Day 1: Fixation, Permeabilization, Blocking, Primary Antibody
4. 3X Washes: Ice-cold PBS for 5 min (Incubate at RT, Orbital shaker)
Wash 1 ロ
Wash 2 ロ
Wash 3 ロ
SECTION: Day 1: Fixation, Permeabilization, Blocking, Primary Antibody
5. Blocking: Add 600uL of 100% Superblocking Buffer. Incubate at RT for 1h with orbital shaker
SECTION: Day 1: Fixation, Permeabilization, Blocking, Primary Antibody
6. 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



SECTION: Day 2: Secondary antibody
7. Remove Primary Ab hybridization buffer. Antibodies can be stored at 4C and re-used. 
SECTION: Day 2: Secondary antibody
8. 3X Washes: Ice-cold PBS for 5 min (Incubate at RT, Orbital shaker)
Wash 1 ロ
Wash 2 ロ
Wash 3 ロ
SECTION: Day 2: Secondary antibody
9. 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

SECTION: Day 2: Secondary antibody
10. 3X Washes: Ice-cold PBS for 5 min (Incubate at RT, Orbital shaker)
Wash 1 ロ
Wash 2 ロ
Wash 3 ロ
SECTION: Day 2: Secondary antibody
11. Stain every well with Hoechst 33342 diluted 1:1000 in PBS. Incubate at RT for 3 min on an orbital shaker
SECTION: Day 2: Secondary antibody
12. 3X Washes: Ice-cold PBS for 5 min (Incubate at RT, Orbital shaker)
Wash 1 ロ
Wash 2 ロ
Wash 3 ロ
SECTION: Day 2: Slides Mounting
13. Remove silicone gaskets from the glass slides.  With a Kimwipe, remove the excess of PBS without touching the surface with the cells


SECTION: Day 2: Slides Mounting
14. 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
SECTION: Day 2: Slides Mounting
15. 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.


SECTION: Day 1: Fixation, Permeabilization, Blocking, Primary Antibody
3. 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