22_SWP_Immunofluorescence_CM

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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
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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:
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Fix cells as above
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Permeablise by incubation with 0.1% TritonX-100 in 1x PBS for 5 minutes
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Wash 3 times with 1x PBS.
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Block with 0.5% BSA in 1x PBS for 30 minutes
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Dilute TRITC or FITC conjugated phalloidin 0.5% BSA (usually 1 in 1000)
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Incubate cells with phalloidin for 15 minutes in the dark
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Wash 3 times with 1x PBS.
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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).
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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
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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.
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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.
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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.
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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.
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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.
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Electrical hazard
If using suction pump during washes, take care with electrical equipment. [Read SWP on using electrical equipment]
List emergency shutdown instructions
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Emergency electrical shutdown button is located in Laboratory
List clean up and waste disposal requirements
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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
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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
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Page 3 of 3
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
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