OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
SOMS GBD SWP 014
School/ Divisional Unit
School of Medical Sciences
Initial Issue date
13-03-2009
Current version
1.0
Current Version
Issue date
20-04-2010
Next review date
20-04-2013
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
Immunohistochemistry
Title:
Description: use specific antibodies to visualize and locate the target signals
Associated risk assessment title and location: RA Handle human tissue 020, RA liquid nitrogen 006, RA using cryostat 007, RA
Waste management 015 located in Room 209B.
Describe the activity or process
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Dissect human tissue in biosafety hood.
Immerse the tissue in Zamboni’s fixative solution and store at 4oC overnight.
Wash the tissue blocks three times in DMSO, three times in PBS and then immerse it in sucrose-buffered saline overnight
at 4oC.
Place the tissue block in tissue TeK bowl, freeze the block within bowl in liquid nitrogen and store the frozen block at –
80oC until cutting procedure start.
Cut sections on cryostat and mount them onto chrome-alum gelatin coated slides.
Clean the sections by three washes in PBS.
Pre-incubate the sections with 3% of H2O2 in PBS for 5-8min.
Incubate the sections with 10% of normal serum for 30min to quench non-specific signals.
Incubate the sections with primary antibody of an appropriate dilution in TBS-TX overnight at room temperature.
Three washes in TBS-TX.
Incubate the sections with secondary antibody diluted to 1:200 in TBS-TX for 2 hours at room temperature.
Three washes in TBS-TX.
Incubate the sections with avidin-biotin complex solution (ABC) diluted to 1:200 in TBS-TX for 1 hour at room temperature.
Three washes in TBS-TX.
Stain the sections with DAB solution for 2-20 min.
Three washes with BPS.
Dehydrate the sections by a series of ethanol (50%, 70%, 95% and 100%) and xylene (three changes) and then mount
the slides with DePeX and coverslip.
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
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Suitable PPE
Slide, one used blade, tissue TeK, fixative, mounting media, cover-slip, ethanol, xylene, chamber for immunoreaction,
pipette, tips and paper towels
Antibodies, ABC solution , DAB solution, H2O2 solution, buffers
Biosafety hood, liquid nitrogen tank and container, chemical fume cupboard, cryostat
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Page 1 of 2
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List potential hazards and risk controls including
specific precautions required
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Potential human sample contamination – all users have been immunized against Hepatitis B, all samples are considered
to be infectious and appropriate PPE is always used, instruments used during procedure are soaked in 70% alcohol, then
rinsed with distilled water after using.
Potential aerosol hazard – wear PPE, use a biosafety hood for dissection. Use chemical fume hood for DAB step.
Injury and contamination from used cryostat blade – Train all users to handle the blade correctly and always dispose the
blade in sharps bin after finished. All users must have immunized against Hepatitis B, wear appropriate PPE during
experiment and go to first aid officer immediately after injury.
Cuts from broken slides – handle the slides carefully and go to first aid officer immediately after injury.
Potential electrical hazard – do annual inspection of electrical equipment and ensure inspection tag is current.
Chemicals may be hazardous – for chemicals check MSDS and wear PPE. Handle DAB in fume hood always, change
gloves when they have touched to DAB and bleach everything which have touched DAB before disposal
List emergency shutdown instructions
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Cryostat has “OFF” switch that can be used in emergency.
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Shut all electrical equipment off at power point in case of emergency.
List clean up and waste disposal requirements
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Clean up the working area, cryostat and instruments with 70% ethanol
Dispose gloves, tips, tubes in biological waste or autoclaved waste bin (human tissue experiment)
Dispose all chemical waste in chemical waste bin
Dispose leftover slides in sharp 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 Labeling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
Australian Standard AS2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
Australian Standard AS2243.7-1991. Safety in laboratories. Part 7: Electrical Aspects.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
Safe Work Procedure Form (OHS026)
UNSW Hazardous Waste Disposal Procedure
Supervisory approval, training, and review
Supervisor: Prof E Burcher
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licensing, training - e.g. course or instruction:
SWP review date: 20-04-2013
Responsibility for SWP review: Fei Shang
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Page 2 of 2
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007