Immunofluorescent Staining of Sialoglycoproteins in Neural Progenitor Cells and Neurons

Abstract

Source: Bai, Q., et al., Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan. J. Vis. Exp. (2019)

This video demonstrates the biotinylation and fluorescent labeling of modified sialoglycoproteins in primary neural stem and progenitor cells, as well as neurons, and their visualization using fluorescence microscopy.

Protocol

1. Immunofluorescent Staining of Sialoglycoproteins in Expanded Primary NSPCs and Differentiated Neurons

1. Prepare BTTAA-CuSO₄ complex 1 30x stock containing 1.5 mM CuSO₄ and 9 mM BTTAA in double-distilled water. Prepare freshly biotin-conjugated buffer 1 containing 50 µM biotin-alkyne, 2.5 mM sodium ascorbate, and 1x BTTAA-CuSO₄ complex in PBS.
2. Remove the inserts from the co-culture plates. Aspirate the culture medium from the bottom wells and wash the neural cells once with pre-warmed PBS.
3. Aspirate the PBS from the wells. Add 1 mL of pre-chilled 4% paraformaldehyde PBS solution per well to the cells and fix them at RT for 10 min. Then, wash the cells 3 times with pre-chilled PBS.
4. Aspirate PBS from the wells and add 1 mL of freshly prepared biotin-conjugated buffer 1 per well into the cells. Incubate the cells at RT for 10 min.
5. Aspirate the reaction buffer from the wells. Wash the cells 3 times with PBS. Prepare the staining buffer containing 1% FBS and 1 µg/mL Alexa Fluor 647-streptavidin. Add 1 mL of staining buffer per well into the cells and incubate the cells at RT for 30 min.
6. Aspirate the staining buffer from the wells and wash cells 3 times with pre-chilled PBS. Prepare the blocking buffer containing 5% BSA and 0.3% non-ionic detergent-100 in PBS. Add 1 mL of blocking buffer per well into the cells and incubate at RT for 10 min.
7. Prepare a primary antibody solution by diluting the anti-nestin and anti-β-tubulin III antibodies together into the blocking buffer at ratios of 1:20 and 1:1,000, respectively. Remove the blocking buffer from the wells and add 1 mL of primary antibody solution per well into the cells. Incubate the cells at 4 °C overnight.
8. Remove the primary antibody solution from the wells. Wash the cells 3 times with pre-chilled PBS. Prepare a secondary antibody solution by diluting Alexa Fluor 488 goat anti-mouse IgG1, Alexa Fluor 546 goat anti-mouse IgG2b, and DAPI together into blocking buffer at a dilution of 1:1,000. Aspirate the PBS from the wells and add 1 mL secondary antibody solution per well into cells. Incubate the cells at RT for 2 h.
9. Aspirate the antibody solution from the wells and wash the cells 3 times with pre-chilled PBS. Afterward, the cells are ready for image capture.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal bovine serum	Gibco	10099141	10%
Paraformaldehyde	Sigma	158127	4%
DAPI	Gibco	62248
6-well plate	Corning	3335
Mouse monoclonal anti-Nestin	Developmental Study Hybridoma Bank	Rat-401	1 to 20
Mouse monoclonal anti-beta-tubulin III	Sigma	T8860	1 to 1000
Alexa Fluor 488 goat anti-mouse IgG1	Invitrogen	A-21121	1 to 1000
Alexa Fluor 546 goat anti-mouse IgG2b	Invitrogen	A-21143	1 to 1000
Triton X-100	Amresco	694
Copper sulfate	Sigma	209198
Alkyne-biotin	Click Chemistry Tools	TA105
BTTAA	Click Chemistry Tools	1236
sodium ascorbate	Sigma	A4034
Alexa Flour 647-conjugated streptavidin	Invitrogen	S21374	1 to 1000