Feb 14, 2023
Immunofluorescence for Primary Brain Cell Cultures
- 1Division of Biology, University of California, San Diego
Protocol Citation: Ashley V Kumar, Francesca Telese 2023. Immunofluorescence for Primary Brain Cell Cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwkzr9vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 27, 2022
Last Modified: February 14, 2023
Protocol Integer ID: 61538
Keywords: immunostaining, immunofluorescence, antibody, immunocytochemistry
Funders Acknowledgement:
NIDA
Grant ID: R21DA056177
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.
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.
Materials
| A | B | C | |
| Name | Company | Catalog | |
| Prolong Glass antifade Mountant | Thermo Fisher Scientific | P36984 | |
| Formaldehyde 16% | Thermo Fisher Scientific | 28906 | |
| Superblocking Buffer 100% | Thermo Fisher Scientific | 37515 | |
| Tween-20 | Millipore Sigma | P1379 | |
| Triton X-100 | Millipore Sigma | T9284 | |
| Hoechst | Thermo Fisher Scientific | H3570 | |
| Poly-l-ornithine: | Millipore Sigma | P3655 | |
| Laminin | Millipore Sigma | L2020 | |
| Poly-D, L-lysine hydrobromide (PDLL) | Millipore Sigma | P9011 | |
| Millicell EZ SLIDE 4-well glass, sterile | Millipore Sigma | PEZGS0416 | |
| Slip-Rite Cover Glass | Thermo Fisher Scientific | 152250 |
BUFFERS:
-Coating buffer in PBS
Poly-D, L-lysine hydrobromide (PDLL) (Sigma #P9011)
Dissolve 5mg in 500ml PBS (0.01mg/ml)
Poly-l-ornithine: 20mg/mL; For usage, dilute to 20μg/mL in ddH2O (1:1000)
Laminin: 1mg/mL; For usage, dilute to 5μg/mL in ddH2O (1:200)
-Fixation buffer
4% Formaldehyde in PBS
- Permeabilization Buffer
0.3% Triton X-100 in PBS
-Antibody hybridization buffer in PBS
10% Superblocking buffer
0.1% Tween-20
-Nuclear staining buffer
Hoechst 33342 1mg/ml solution in water
Day 1: Fixation, Permeabilization, Blocking, Primary Antibody
Day 1: Fixation, Permeabilization, Blocking, Primary Antibody
3h 30m
3h 30m
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
Day 2: Secondary antibody
2h
2h
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
Day 2: Slides Mounting
30m
30m
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.