Application
Immunocytochemistry
tPA-FITC ab92679 was tested in an in vitro uptake assay performed on astrocytes isolated from P2-P3 mouse pups. The astrocytes were cultured in 4 well plates coated with PDL at a density of 25.000 cells/well in DMEM, 10% horse serum and 1% antibiotica (P/S).
Before tPA application, the astrocytes were washed 2 times with pre-warmed DMEM to wash out the horse serum.
After the washes, 140 nM tPA-FITC in DMEM was applied for 15 minutes at 37 degrees. Afterwards, the cells were washed 2 times with 1xPBS and subsequently fixed with 4% PFA at room temperature.
For the ICC, after fixation, the cells were washed 2 times with 1xPBS, blocked with 1xPBS-Triton-X containing 10% NGS (goat serum) and incubated with a GFAP antibody (to visualise the astrocytes) for 1 hour at room temperature. Afterwards, the cells were washed 2 times with 1xPBS and the secondary antibody was applied together with DAPI to visualise the cell nuclei and incubated for 30 min at room temperature in the dark. After washing 3 times with 1xPBS, embedding medium was applied.
For control conditions, some cells were incubated with 140 nM tPA-FITC in DMEM for 15 minutes at 4 degrees (cells do not uptake tPA in these conditions) and some cells with 140 nM Natural Human Serum Albumin protein-FITC (ab8030) in DMEM for 15 minutes at 37 degrees.
The uploaded image shows wild type mouse astrocytes incubated with 140nM tPA for 15 minutes at 37 degrees. Scale bar is 20µm.
Before tPA application, the astrocytes were washed 2 times with pre-warmed DMEM to wash out the horse serum.
After the washes, 140 nM tPA-FITC in DMEM was applied for 15 minutes at 37 degrees. Afterwards, the cells were washed 2 times with 1xPBS and subsequently fixed with 4% PFA at room temperature.
For the ICC, after fixation, the cells were washed 2 times with 1xPBS, blocked with 1xPBS-Triton-X containing 10% NGS (goat serum) and incubated with a GFAP antibody (to visualise the astrocytes) for 1 hour at room temperature. Afterwards, the cells were washed 2 times with 1xPBS and the secondary antibody was applied together with DAPI to visualise the cell nuclei and incubated for 30 min at room temperature in the dark. After washing 3 times with 1xPBS, embedding medium was applied.
For control conditions, some cells were incubated with 140 nM tPA-FITC in DMEM for 15 minutes at 4 degrees (cells do not uptake tPA in these conditions) and some cells with 140 nM Natural Human Serum Albumin protein-FITC (ab8030) in DMEM for 15 minutes at 37 degrees.
The uploaded image shows wild type mouse astrocytes incubated with 140nM tPA for 15 minutes at 37 degrees. Scale bar is 20µm.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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提交于 May 09 2017