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ELISpot plate coating procedure1. Pretreat with ethanol
Add ethanol into each well and the membrane will turn into gray after contact with ethanol, which indicate that PVDF has been activated. If you coat several plates at one time, we recommend treating with 1-2 plates and continue with the next step immediately.
The ethanol concentration and treating time is very critical:
MSIP and IPEL plate--Add 15 µl 35% ethanol into each well and do not stay more than 1 min.
MAIPSWU plate--Add 50 µl 70% ethanol into each well and do not stay more than 2 min.
2. Wash with sterile water
Empty the plate and wash it with 200 µl each well with sterile water for 5 times. If you use MSIP or IPFL plate, you may add the water into wells without empty the plate.
3. Add the coating antibodies
Empty the plate and add 100µl PBS into each well to dilute the antibodies. We recommend the concentration at 10-15 µg/ml (1-1.5 µg antibodies each well). Stay at 4℃ for a night.