1.0 1237086432018 OMERO.editor PROTOCOL_ARCHIVE Immunostaining This protocol has been adapted from the Lamond Lab protocol found here: http://www.lamondlab.com/f7protocols.htm Fix Fix cells in [[0]]. Permeabilize with 1% Triton X-100 in PBS for 15 minutes at room temperature. Block by incubating cells for 10 min in blocking buffer. Perform all blocking and antibody incubation steps in a humidified chamber. fixative 0 OPTIONAL ENUMERATION Paraformaldehyde Methanol Paraformaldehyde SINGLE_STEP Stain STEP_GROUP Primary Antibody Incubate cells with [[1]] diluted [[2]] in blocking buffer for [[3]]. If you have never used the antibody before, try several dilutions (low of 1:100 to high of 1:1000, for example). Wash coverslips 3 X 10 minute with PBS. antibody name 1 OPTIONAL TEXT dilution 2 OPTIONAL ENUMERATION 1:100 1:200 1:250 1:500 1:1000 1:2000 incubation time 3 OPTIONAL NUMERIC minutes SINGLE_STEP Secondary Antibody Incubate cells with [[4]] diluted [[5]] in blocking buffer for [[6]]. Wash coverslips 3 X 10 minute with PBS. antibody name 4 OPTIONAL TEXT dilution 5 OPTIONAL ENUMERATION 1:100 1:200 1:250 1:500 1:1000 1:2000 incubation time 6 OPTIONAL NUMERIC minutes SINGLE_STEP Mount If desired, stain DNA with DAPI [[7]] (1:15,000 dilution in water of a 5 mg/ml stock). A good RNA stain, which fluoresces in the red channel, is Pyronin Y (1:10,000 dilution in water of a 10 mM stock; must stain with DAPI first). Wash well with PBS and mount cells as desired (we use either MOWIOL/Dabco, which hardens, or the liquid mounting media Vectashield from Vector Labs, in which case the coverslips must be sealed with nail varnish). Slides can be stored in the fridge or freezer until analyzed by microscopy. DAPI stain 7 OPTIONAL ENUMERATION true true false SINGLE_STEP