Fixation and Immunostaining protocol

Prepare 1x Fix by dilution from 2x Fix (see Description)

Gently aspirate culture supernatant, wash gently with PBS, and replace with cold Fix buffer

Incubate 30min @ 4° C or on ice

Aspirate fix and wash gently 3x with PBS (incubate 1-2min each)

Aspirate  and add Quench to permeabilize cells/tissue, at least 15 min room temperature (RT). Note: this and all steps in humidified chamber (HC). Can store samples for weeks/months at 4 degrees

Aspirate and add Block buffer, 30 min RT

Incubate with primary antibodies cocktail (1-2 hrs RT or 4°C overnight (ON)

Wash 3x  5min (wash buffer)

Prepare secondary antibodies in Wash buffer (1:500-1:1000), filter, light protect (LP).Note: This and all subsequent steps LP

Incubate with secondary antibodies 1hr RT, light protected (LP)

Wash 3x 5min

Incubate with 1x DAPI in Wash buffer 5-10 min

Wash 1x

Rinse very briefly with filtered H2O, 2x

Remove excess water, do not dry samples

Add Fluoromount-G (~20ul/well or ~100ul/slide) and apply No 1.5H coverslip (slowly lower, no bubbles) or other mountant

Dry ON, LP, RT