Immunofluroescence - Methanol Fixation Protocol 1X PBS 20ºC Freezer- dH2O Pre-chilled Methanol Stored at -20ºC P20 Tips .Seed cells onto round coverslips in 24-well plates .1 1.Allow cells to grow for at least 24h, ideally 48h .2 Aspirator For use with aspirator .Change media after 24h to remove cell debris/dead cells .3 If an experimental treatment is needed prior to fixation (drug treatment, .4 .irradiation, etc.), proceed with the treatment before the next step .Use a new tip and aspirate to carefully remove media from each sample .5 2.Gently add PBS to each sample to rinse .6 3.Using a new tip, aspirate to remove PBS from each sample .7 4.Quickly add about 200uL of -20ºC methanol to each sample .8 5.Incubate samples in -20ºC freezer for 3 to 5 minutes, BUT NOT ANY LONGER .9 Remove the samples from the freezer and, using a new tip, quickly aspirate the .10 6.methanol from every sample .Allow the samples to air dry for 2 minutes at room temperature .11 .Add dH 2 O to each sample and allow to re-hydrate for 2 minutes .12 1 The goal is to have cells at 50-70% confluence before fixation. 2 200uL of PBS should be sufficient. 3 This aspirated media represents biohazardous waste, which must be disposed of as biohazardous waste. Ensure that if there is any biohazardous waste, and it's treated with bleach, that this waste+bleach DOES NOT MIX with any alcohol component, as this can create toxic and dangerous by-products. 4 It is important to work through this QUICKLY, because the fixation begins as soon as the methanol is added to the samples. If fixing 2 plates at a time, ideally fix one plate (methanol, incubation, air dry, rehydrate, rinsed with PBS) before proceeding to the next one. 5 Some protocols include an incubation that is up to 10-15 minutes. Alcohol-based fixation permeabilizes cells but also dehydrates the cells and can lead to distorted cell morphology or even failed IF stainings. 6 This waste represents toxic, flammable waste (specifically for the methanol), and must be disposed of separately in the flammable waste. It ab- solutely CANNOT go down the drain. It may be possible to substitute methanol for ethanol to reduce the risk from the waste, but the protocol may change slightly.
. Aspirate to carefully remove the dH 2O .13 .Rinse samples with 1X PBS .14 .Repeat for a total of 3 PBS rinses .15 .Proceed to standard immunofluorescence protocol .16