OHS026 Safe Work Procedure Faculty/Division Medicine Document number SOMS.CGM.SWP022 School/ Divisional Unit SoMS, Oncology Research Unit/MDU Initial Issue date 30/06/09 Current version 1.0 Current Version Issue date 30/06/09 Next review date 30/06/12 The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this form. Safe Work Procedure Title and basic description Title: Immunofluorescent staining of cells with antibodies and phalloidin Description: To safely and correctly fix, permeablise and immunofluorescently stain cells with antibodies and phalloidin Associated risk assessment title and location:22_RA_Immunofluorescence_CM Describe the activity or process Wash cells on coverslips (or in chamber slides) with 1x PBS Fix cells by incubation with 4% paraformaldehyde in 1x PBS for 15 minutes (use 2mL per well in 6-well plate, adjust volume as appropriate for other well sizes). Wash 3 times with 1x PBS. Permeablise cells by incubation with cold methanol (stored at -80°C) for 15 minutes Wash 3 times with 1x PBS. Block with 2% FBS in 1x PBS for 30 minutes Dilute primary antibodies to appropriate dilution in 2% FBS (usually between 1 in 50 and 1 in 1000) Incubate cells with primary antibodies for 2h at room temperature, or overnight at 4°C (use 200μL per coverslip) (NB: dilutions and incubation periods will vary depending on the particular antibody used) Wash 3 times with 1x PBS. Dilute fluorescent secondary antibodies to appropriate dilution in 2% FBS (usually 1 in 1000) Incubate cells with secondary antibodies for 1h at room temperature (NB: cells should be kept in the dark as much as possible from this point onwards) Wash 3 times with 1x PBS. Dilute DAPI nuclear stain in 2% FBS (1 in 10 000) Incubate cells with DAPI for 1-5min Wash 3 times with 1x PBS. Mount coverslips onto microscope slides using FluorSave reagent (for chamber slides, mount coverslip over cells on slide) Seal edges of coverslip with nailpolish For phalloidin staining: Fix cells as above Permeablise by incubation with 0.1% TritonX-100 in 1x PBS for 5 minutes Wash 3 times with 1x PBS. Block with 0.5% BSA in 1x PBS for 30 minutes Dilute TRITC or FITC conjugated phalloidin 0.5% BSA (usually 1 in 1000) Incubate cells with phalloidin for 15 minutes in the dark Wash 3 times with 1x PBS. Mount as above NOTE: This is a general protocol. The precise protocol for fixation, permeablisation and staining is dependent on the individual antibodies and cells being used. Specific instructuction for different antibodies can be found in manufacturers specifications). ___________________________________________________________________________________________________________ ___________ Page 1 of 3 Safe Work Procedure Date Effective: 01/01/2007 Uncontrolled document when printed Current Version: 1.2, 15/08/2007 List all resources required including plant, chemicals, personal protective clothing and equipment, etc Cells grown on coverslips (or in chamber slides) 1xPBS 1xPBS with 2% FBS 1xPBS with 0.5% BSA Primary antibodies Secondary fluorescent antibodies TRITC and FITC conjugated phalloidin DAPI Glass microscopy slides Fluorsave mounting reagent List potential hazards and risk controls including specific precautions required Read MSDS of all chemicals before use. Exposure to paraformaldehyde Experiments with animals suggest that this material may act as a carcinogen. Ingestion may be fatal. May cause heritable genetic damage. May cause irreversible damage to sight. Corrosive. May cause skin or eye burns or irritation. Can cause severe burning of mouth and stomach. Harmful by inhalation, ingestion or skin absorption. Wear PPE (gloves, gown and closed shoes). Wear goggles where splashing is possible. Exposure to methanol Toxic by inhalation, ingestion or skin absorption. May be a reproductive hazard. Ingestion may be fatal. Risk of very serious, irreversible damage if swallowed. Exposure may cause eye, kidney, heart and liver damage. Chronic or substantial acute exposure may cause serious eye damage, including blindness. Irritant. Narcotic. Wear PPE (gloves, gown and closed shoes). Wear goggles where splashing is possible. May react violently with acids, acid chlorides, acid anhydrides, oxidizing agents, reducing agents and alkali metals. Protect from moisture. Highly flammable – keep away from open flame and ignition sources. Exposure to DAPI May be harmful is swallowed, inhaled or absorbed through skin. Causes eye irritation. Avoid contact with eyes, skin, and clothing. Avoid prolonged or repeated exposure. Keep tightly closed and avoid light. Wear PPE (gloves, gown and closed shoes). Wear goggles where splashing is possible. Exposure to phalloidin Very toxic. May be fatal if swallowed, inhaled or absorbed through the skin. Wear PPE (gloves, gown and closed shoes). Wear goggles where splashing is possible. Electrical hazard If using suction pump during washes, take care with electrical equipment. [Read SWP on using electrical equipment] List emergency shutdown instructions Emergency electrical shutdown button is located in Laboratory List clean up and waste disposal requirements Dispose of all liquid waste that has had contact with cells in appropriate biological waste containers Dispose of methanol in dedicated methanol waste bin. Dispose of phalloidin waste in toxic waste bin Dispose of glass coverslips and slides in sharps bin List legislation, standards and codes of practice used in the development of the SWP NSW OHS Act 2000 NSW OHS Regulation 2001 Australia Dangerous Goods Code Code of Practice for the Labelling of Workplace Substances AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance AS/NZS 1336:1997 Recommended Practices for Occupational Eye Protection UNSW Hazardous Waste Disposal Procedure ___________________________________________________________________________________________________________ ___________ Page 2 of 3 Safe Work Procedure Date Effective: 01/01/2007 Uncontrolled document when printed Current Version: 1.2, 15/08/2007 Supervisory approval, training, and review Supervisor: Peter Gunning Signature: Plant custodian: Signature List competency required – qualifications, certificates, licencing, training - eg course or instruction: Training as per Training Needs Analysis, Induction to Lab, Training in this SWP ___________________________________________________________________________________________________________ ___________ Page 3 of 3 Safe Work Procedure Date Effective: 01/01/2007 Uncontrolled document when printed Current Version: 1.2, 15/08/2007