Hello,
I am planning to do a cytokine immunoflourescence in spleen tissue slices. In bibliography most methods do not permeabilize cells, thereby detecting extracellular protein (associated to cell membrane or matrix). In consequence it is impossible to determine which is the source cell.
Moreover, in flow cytometry protocols there is a stimulation / traffic blocking previous step that is supposed to enhance the cytokine signal (usually PMA, Ionomicin, LPS, Brefeldin, Monensin).
I was wondering if there is some kind of adaptation of the former protocol for tissue slides.
Thanks for reading,
Martin
I label IL-6 in rat brain slices by immunofluorescence. I attach a photo of a dual labeled slice. The free floating slice of brain tissue (of 20 micrometer thickness) was blocked in 10% BSA and 5% donkey serum, incubated in coctail of primary antibodies (mouse anti-IL6 from Abcam and rabbit anti c-Fos from Santa Cruz in dilution 1:500). Secondary antibodies were donkey Alexa Fluor 546 anti-mouse, and donkey Alexa Fluor 488 anti rabbit. The effect is that IL-6 is orange, c-Fos is green. I noticed that IL-6 is detected rather on the membrane of the cells, as orange, hollow circles appear.
Hello
Look at this question which discussed the same issues :
https://www.researchgate.net/post/Has_anyone_had_success_with_IHC_for_inflammatory_cytokines_in_mice_brains
And What about this article?
Article Role of biphasic changes in splenic dendritic cell activity ...
Good Luck
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