I'm testing a protocol for viral quantification by IF; it is being standarizing from the fixation step, as a fixative choosen are Methanol (10mins) and 4% Formaldehyde-10%Methanol (15mins) (Is hard to buy parafolmadehyde in my country) for PK15 cells in 96-well culture plates; The permeabilization solution of cell for formaldehyde treated cells is made with 0.1% Triton X-100/PBS (15mins), and the blocking solution for both assays are made with 4% BSA/PBS (30mins). I'm testing two staining solution to dilute the antibody (FICT conjugated Anti-CSFV monoclonal Antibody - BIO 272): 1) 2% BSA/PBS + Ab; and, 2) 0.3%BSA/0.05% Tween 20/PBS + Ab; both incubated overnight. However, the result I get for both experiments is in my opinion are inespecific fluorescence, because it is shown in my negatives control; additionally, the fluorescence stronger signal are in dead cell conglomerates, except for methanol treated cells which shows a very week signal for negative control and samples. I share with you some pictures I took. What suggestion can be made according to this experience? Could you share a protocol for immunofluorescence of CSFV in PK15 cells? Is the Antibody not working?
Please look at the following below links which may help you in your analysis:
https://www.biorxiv.org/content/biorxiv/early/2018/08/10/389361.full.pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719638/pdf/zjm2710.pdf
Thanks
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