To begin, seed HEK293A cells on gloss cover slips placed into a 24 well plate containing high glucose DMEM until they become 80%confluent. The next day, starved the cells for two hours in Earl's balanced salt solution. After treatment, aspirate the medium and add 500 microliters or 4%formaldehyde solution in PBS and incubate for 20 minutes at room temperature to fix the cells.
After fixation, replace the formaldehyde solution with 500 microliters of PBS. Next, quench the free aldehyde group by adding 500 microliters of 50 millimolar ammonium chloride solution in PBS for 10 minutes at room temperature. Replace the ammonium chloride solution with 500 microliters of a 50 microgram per milliliter digitonin solution in PBS for five minutes to permeabilize the cells.
After permeabilization, replace the digitonin solution with 500 microliters of PBS repeating PBS washing three times. After the last wash, incubate the cells in 500 microliters of blocking solution for 30 minutes at room temperature. After incubation, replace the blocking solution with 500 microliters of PBS.
Next using tweezers, collect the cover slips from the well and remove the excess PBS using thin tissue wipes. In the humidified chamber, gently lay down the cover slip with cell side down onto a 50 microliter drop of primary antibody solution. Incubate the cells in a humidified chamber for one hour at room temperature.
After incubation, collect the cover slips and drain off the excess primary antibody solution. Place the cover slips back in the 24 well plate with the cell side up and wash them three times with PBS. After the last wash, collect the cover slips and drain off the excess PBS before placing each cover slip with cell side down onto a 50 microliter drop of secondary antibody solution.
Incubate in a humidified chamber for one hour. After incubation, drain off the excess secondary antibody solution and wash the cover slip three times with 500 microliters of PBS in the 24 well plate. After draining excess PBS, place each cover slip with cell side down onto a 50 microliter drop of hooks solution diluted one to 4, 000 in PBS in a humidified chamber.
Incubate in a humidified chamber for five minutes. Next, remove the excess hooks solution and wash the cover slips three times with PBS and once with deionized water in a 24 well plate. After draining excess deionized water, place each cover slip onto a 10 to 20 microliter drop of mounting solution spotted onto a microscope slide avoiding the formation of air bubbles.
