Hi everybody, I'm trying to obtain microglia purification from rat pupets from 1-3 days. After having the glia culture for two weeks, I tried to purify the microglia following this protocol https://bio-protocol.org/e314.

To assure I have only microglia cells I performed an immunofluoresnce assay with the following protocol.

1. Retire cell medium

2. Wash with PBSx2

3. Fix cells with PFA 4%

4. Wash with PBSx3

5. Blocking and permebilization with BSA0,1%, Triton 0,1% in PBS for 2h, RT

6. Wash with PBSx3

7. Incubate primary antibodys (rabbit polyclonal Iba1, synaptic systems # 234 003 and goat polyclonal GFAP, Abcam # ab53554) with BSA0,1%, Triton 0,1% in PBS, ON at 4ºC

8.Wash with PBSx3

9. Incubate with secondary antibodys (Alexa 488 anti rabbit, Alexa 555 Anti goat), with BSA0,1%, Triton 0,1% in PBS fo 2h, RT

10.Wash with PBSx3

11. Incubate con DAPI

12. Mounted and coverslipped

The images I obtained with the microscope are the following. (A) In the green lut I cannot see specificty for Iba1+ cells, since I see also astrocytes. How can I improve the specicifity for this antibody? Should I perfomer the IF with order antibody to mark microglia cells? In the red lut I can also see some Iba1 cells but I think that this is autofluorescence, am I okey? (B) Microglia cells in brieghtfield. (C) Glia mixed ulture in brieghtfiled before purification. The images obatined in brightfield (B) are microglia cells for sure?

Thank you! :)