Korea University
Question
Asked 10th Oct, 2017
How to avoid the nonspecific staining for Tuj1 in the DIV9 hippocampal neurons isolated from prenatal mice?
Hippocampus was isolated from ICR mice embryos at embryonic Day 18.5 using Ca2+ and Mg2+ free Hanks' balanced salt solution. Isolated hippocampus were trypsinized and triturated with fire-polished Pasteur pipettes, then strained through 40-μm nylon mesh. These cells were cultured in Neurobasal medium with 2% B27 supplement, 0.5 mM L-glutamine, 25 μM L-Glutamate, and 1% antibiotics mixture. The one half of the same culture media without Glutamate or antibiotics was used for media changes after 4d.
IF staining: The hippocampal neurons were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature. Fixed neurons were blocked with 5% bovine serum albumin and then incubated with primary antibody against Tuj1 diluted in 1%BSA/PBS for 1 h at room temperature. Followed by staining with Secondary fluorescent conjugated antibody diluted in 1% BSA/PBS. Between each step, PBS washing was done three times.
All Answers (3)
Korea University
Hi, Renuka.
The staining looks quite specific. However, you have lots of spots on background that may be cell fragments surrounding neurons, probably derived from cell isolation procedure. They can be eliminated by differential cell separation by simple spinning down three times each in 10ml PBS volume in 13 ml Falcon tube make the size fractionation easier at low speed emperically. Or First collect cell clumps in the 10ml dish suspension under a steromicroscope, and gentle 2~3 whirlings would make them gather in the center, and collect them by a pasteur pipette. You could even separate single cells from small spotty cell fragments (they are membrane-bound containing TuJ1 in themselves showing intensive staining and attached firmly on the coated-ECM on coverslip). If you mix them completely by upside-down the tube, followed by gentle whirling in 10ml dish. cell fragment would not gather to the center, but remains periphery of the dish. Collect single cell in the center similarly. Repeat this after spining everything down, resuspending in 10ml, then dish. You will get clean neurons.
Spin every diluted antibody sol and 5% BSA sol at highest rate to remove particles, and use primary ab more diluted than you are using now. 0.01% Tween will also help a dark bark ground in 20(v/v) in PBS+BSA.(PBT-most people use it).
Best wishes.
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