小中大[求助]关于细胞流式的同型对照的问题？
关于细胞流式的同型对照的问题？
在做foxp3流式，看到说明书上说到同型对照，
1. Add 100 µl of prepared cells (1x106) to each tube.
2. Stain surface molecules such as CD4, CD8, CD25, etc. following the Surface Staining Protocol (cuturl('http://www.ebioscience.com/ebioscience/appls/FCS.htm')).
3. Wash in cold Flow Cytometry Staining Buffer (or cold PBS).
4. Resuspend cell pellet with pulse vortex and add 1 ml of freshly prepared Fixation/Permeabilization working solution to each sample. Pulse vortex again.
5. Incubate at 4°C for 30 - 60 minutes in the dark.
6. Wash once by adding 2 ml 1X Permeabilization Buffer (made from 10X Permeabilization Buffer) followed by centrifugation and decanting of supernatant.
7. 【OPTIONAL】 Block with 2% (2 µl) normal rat serum in 1X Permeabilization Buffer, in approximately 100 µl volume, at 4°C for 15 minutes.
8. Without washing after blocking step, add 20 µl fluorochrome conjugated anti-human Foxp3 antibody or isotype control in 1X Permeabilization Buffer and incubate at 4°C for at least 30 minutes in the dark. Please perform further titration for optimal staining in your own assay system.
9. Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
10. Repeat step 9.
11. Resuspend in appropriate volume Flow Cytometry Staining Buffer and analyze on cytometer. Please note that due to the fixation and permeabilization procedure, the FSC/SSC distribution of the cell population will be different than live cells. Therefore the gate and voltages will need to be modified.
其中第7部中正常大鼠血清是直接从大鼠血清中离心获得吗？还是有特异型成品啊？请教做过相关实验的人，拜求！
