Quantification of Filamentous Actin Puncta in Rat Cortical Neurons

Abstract

Source: Li, H., et al. Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons. J. Vis. Exp. (2016)

This video demonstrates the procedure for fluorescently staining rat cortical neurons for microscopy. It targets F-actin and dendritic proteins to analyze structural integrity and calculate F-actin density, which is crucial for studying neuronal function and health.

Protocol

1. Fluorescent Labeling and Immunocytochemistry

Note: The immunofluorescent labeling of primary cortical cell cultures was carried out in glass bottom 35 mm cell culture dishes with a working volume of 1 ml.

1. Wash the glass-bottom dish twice with phosphate-buffered saline pH 7.4 (PBS).
2. Fix cells with 4% paraformaldehyde for 15 min at room temperature (RT).
3. Wash cells twice with PBS and permeabilize with 0.1% Triton X-100 in PBS for 5 min.
4. Treat cells for 20 min at RT with an F(filamentous)-actin-specific stain, AlexaFluor 488 Phalloidin (1:40, 25 µl of Phalloidin in 1 ml PBS).
5. Rinse cells twice with PBS and block with 10% normal goat serum at RT for 1-2 hr.
6. Dilute the chicken polyclonal anti-MAP2 (microtubule-associated protein 2) antibody (1:2,500) in PBS with 2% normal goat serum and incubate O/N (overnight) at 4 ⁰C.
7. Following incubation, rinse in PBS twice.
8. Dilute the secondary antibody (Alexa Red 594-conjugated goat anti-chicken IgG (1:500) with 2% normal goat serum and incubate for 2 hr at RT.
9. Rinse with PBS and add 10 µl/dish of Hoescht dye.
10. Incubate for 3 min at RT and wash with PBS twice.
    Note: The labeled cells are now ready for step 2 (F-actin puncta counting).
11. Preserve the cell sample with 100 µl of antifade reagent as a coverslipping agent and keep it at 4 ⁰C in the dark for long-term storage.
    Note: Phalloidin is relatively stable in PBS at +4 °C in the dark for up to 1 week, and fluorescence can be preserved with antifade coverslipping reagents. However, optimal images are obtained from 1-3 days after staining since Phalloidin may start to diffuse out with extended storage.

2. F-actin Puncta Counting

1. Turn on the fluorescent microscope and switch to 20× objective. Open microscope software and set up program at 1280×960 pixel image size and 0.17 µm/pixel image resolution at 1× zoom.
2. Acquire images of co-labeled F-actin/MAP2 neurons under green (495 nm) or red (613 nm) fluorescent channels.
3. Choose 5 Green (F-actin)/Red (MAP2) immunolabeled/Blue (Hoescht) fluorescent images of individual neurons with clearly defined dendritic arbors.
4. Identify the F-actin-rich structures in the second-order dendritic segment (length range 25-75 µm) with continuous MAP2 immunofluorescence.
5. Rotate the selected region of images to a horizontal level. Copy and paste as a new image.
6. Subtract the background of images, using a consistent adjustment for each dish.
7. Count the bright green F-actin puncta and trace the length of the selected dendritic segment manually by trained independent observers. Export the data to a spreadsheet file.
8. Calculate the density by dividing the total F-actin labeled puncta (N) by the length (L) of the MAP2 labeled dendrites. Express data as the number of F-actin puncta per 10 µm of dendrite
   Note: Puncta (size ≤1.5 µm) of F-actin fluorescence with a peak intensity of at least 50% above the average intensity of staining in the dendritic shaft were included in each selected dendritic segment.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm Glass Bottom Dishes No. 1.5 coverglass	MatTek Corporation	P35G-1.5-20-C
DMEM/F12 medium	Life Technologies	10565-018
Poly-L-Lysine	Sigma	P9155
Paraformaldehyde	Sigma	P6148
AlexaFluor 488 Phalloidin	Life Technologies	A12379
Normal horse serum	Life Technologies	26050-070
Chicken polyclonal anti-MAP2	abcam	Ab92434
Alexa Red 594-conjugated goat anti-chicken IgG	Life Technologies	A11042
NucBlue Live cell stain ReadyProbes Reagent Hoescht 33342	Life Technologies	R37605
NIS-Elements software package	Nikon Instruments
Nikon Eclipse TE2000-E inverted fluorescent computer-controlled microscope	Nikon Instruments