THE UNIVERSITY OF NEWCASTLE- SCHOOL OF BIOMEDICAL SCIENCES ASHMAN LABORATORY

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1 Page: 1 of 8 1. Risk Assessment: This Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this procedure. Therefore, personnel are requested to conduct their own Risk Assessment before using this procedure to include any extra hazards introduced by the task performed. TASK PERFORMED To perform an immunohistochemistry stain on mounted sections HAZARDS 1. Chemical hazard Methanol FLAMMABLE, TOXIC Risk phrases: R11 highly flammable R23/24/25 toxic by inhalation, in contact with skin and if swallowed R39/23/24/25 Toxic: danger of very serous irreversible effects through inhalation, in contact with skin and if swallowed. 2. Chemical hazard - Hydrogen Peroxide OXIDISING AGENT, CORROSIVE Risk phrases: R5 Heating may cause an explosion R8 Contact with combustible material may cause fire R20/22 Harmful by inhalation and if swallowed R35 Causes severe burns R41 Risk of serious damage to eyes 3. Chemical hazard DAB HARMFUL, POSSIBLE CARCINOGEN Risk phrases: R22 - Harmful if swallowed (limited evidence) R34 Danger of cumulative effects R62 Possible risk of impaired fertility 4. Chemical and flammable hazard fixative (acetone) FLAMMABLE Risk phrases: R11 Highly flammable R36 Irritating to eyes WRITTEN BY CHECKED BY AUTHORIZED BY NAME (signed) Ben Copeland Lauren Watt SBS&P Safety Officer DATE 26th October October May 2011 Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of 8 R65 HARMFUL may cause lung damage if swallowed R66 Repeated exposure may cause skin dryness and cracking R67- Vapours may cause drowsiness or dizziness 5. Chemical hazard xylene FLAMMABLE Risk phrases: R10 - Flammable R20/21 harmful by inhalation and in contact with skin R36/38 Irritating to eyes and skin R52 Harmful to aquatic organisms R65 HARMFUL - may cause lung damage if swallowed R67 Vapours may cause drowsiness and dizziness 6. Chemical and flammable hazard R36/38 - Vectamount NOT HAZARDOUS (according to manufacturer) 7. Chemical hazard Lillie Mayer Haemotoxylin IRRITANT Risk phrases: R36/38 Irritating to eyes and skin 8. Flammable hazard graded ethanol (50%, 70% and 100%) FLAMMABLE Risk phrases: R11 Highly flammable R36 Irritating to eyes 9. Burns / scalds from use of heating block and microwave 10. Sharps hazard broken or chipped microscope slides RISK ASSESSMENT 1. Risks are considered very low for all hazards if the risk controls are followed RISK CONTROL 1. Methanol safety phrases: S01 keep locked up S16 keep away from sources of ignition. No smoking. S23 do not breather gas/fumes/vapour/spray S25 avoid contact with eyes 2. Hydrogen peroxide safety phrases S1 - Keep locked up

3 Page: 3 of 8 S17 Keep away from combustible materials S23 Do not breathe gas/fumes/spray S25 Avoid contact with eyes 3. DAB safety phrases S13 Keep away from food, drink and animal feedstuffs S27 Take off immediately all contaminated clothing S40 To clean the floor and all objects contaminated by this material use water and detergent S53 Avoid exposure obtain special instructions before use 4. Use Diaminobenzidine in fume hood. 5. Fixative (acetone) safety phrases S16 Keep away from sources of ignition. No smoking. S23 Do not breathe gas/fumes/vapour/spray S25 Avoid contact with eyes S36 Wear suitable protective clothing 6. Use acetone in fume hood. 7. Xylene safety phrases S23 Do not breathe gas/fumes/vapour/spray S51 Use only in well ventilated areas S9 Keep container in a well ventilated place S53 Avoid exposure obtain special instructions before use 8. Use xylene in fume hood. Wear NITRILE gloves 9. Xylene should be stored and handled in glass jars and containers as it may degrade some plastics. 10. Use mountant in fume hood. Wear NITRILE gloves 11. Lillie Mayer Haemotoxylin - safety phrases S23 Do not breathe gas/fumes/vapour/spray S24 Avoid contact with skin S39 Wear eye/face protection S40 To clean the floor and all objects contaminated by this material use water 12. Ethanol safety phrases S23 Do not breathe gas/fumes/vapour/spray

4 Page: 4 of 8 S39 Wear eye/face protection S51 Use only in well-ventilated areas S9 Keep container in a well ventilated place 13. Keep ethanols, xylene acetone and mountant away from ignition sources. 14. Allow solutions to cool for 20 minutes, prior to removing from microwave. 15. Use thermal protective gloves when handling heated materials. 16. Check all slides for chips or cracks before use and discard any suspect slides into Sharps container. 17. Do not force slides into containers. 18. Appropriate training should be provided from qualified persons experienced in this procedure 19. Wear appropriate personal protective equipment - lab coat, safety glasses and gloves are to be worn at all times. 2. Purpose: 2.1. To perform an immunohistochemistry stain on sectioned tissues (frozen fixed and paraffin embedded) 3. Equipment: 3.1. Humidified chamber 3.2. "Pap pen" or a paraffin bead 3.3. Staining racks 3.4. Staining dish 3.5. Inverter microwave 4. Materials: 4.1. Microscope slides 4.2. Paraffin embedded mounted sections 4.3. Sections known to stain with Primary Antibody (Positive Control) 5. Reagents: M PBS

5 Page: 5 of Blocking Serum - (available within an ABC kit from Vector laboratories cat # VEPK-6102) 5.3. Antibody diluent (0.1M PBS/0.3% Triton X) 5.4. Citrate buffer (available from Vector laboratories) 5.5. Quenching Solution - Methanol:dH 2 O (1:1) with 3% H 2 O Primary antibody 5.7. Biotinylated Secondary Antibody (available within an ABC kit from Vector laboratories cat # VEPK-6102) 5.8. Avidin - Biotin Horseradish Peroxidase complex (available within an ABC kit from Vector laboratories cat # VEPK-6102) 5.9. Steptavidin/biotin blocking kit (available from Vector laboratories cat # VESP- 2002) Diaminobenzidine tetrahydrochloride (DAB) (Available from Vector laboratories in premixed dropper bottles, cat # VESK-4100) Note this procedure only covers premixed DAB reagent. DAB in powder form should NOT be used in this procedure Lillie-Mayer Haematoxylin % Ethanol % Ethanol % Ethanol Xylene clearing solution Vectamount permanent mounting medium (available from Vector laboratories cat # VEH-5000) 6. Set Up: 6.1. Ensure that you have read and understood the Safety Precautions (Section 7 of this SOP) prior to commencing this procedure. 7. Safety Precautions: 7.1. Good laboratory techniques are to be used at all times Always use nitrile gloves when using xylene and mounting media. These chemicals dissolve latex.

6 Page: 6 of Always use xylene in fume cupboard Always use alcohols in fume cupboard and keep away from ignition sources 8. STAINING PROTOCOL ALL STEPS ARE DONE AT ROOM TEMP UNLESS STATED OTHERWISE Day Heat the slides on the heating block for ~2 hrs at 50 o C to melt the paraffin and ensure complete removal. Paraffin Embedded Sections (already mounted) use designated slide basket and slide troughs in fume hood (alternatively coplin jars may be used) Dewax the slides in 2 x 5 xylene 8.3. Rehydrate in 2 x 5 100% EtOH 8.4. Wash slides 2 x 5 in PBS 8.5. Quench endogenous peroxidase activity with 2 ml H 2 O 2 in 200 ml PBS for 5 at RT x 5 PBS. Make up 500 ml citrate buffer for microwaving Microwave slides; fill two racks (24 slides/rack) with blank slides. Put racks into pots and fill to the marked line (250 ml). Remove blank slides as needed and replace with sections to be microwaved. Put the lids on, secure with a rubber band and put into microwave. Microwave slides for 3 on high to boil and then 15 on 3. Wait 2o minutes and then remove pots from the microwave and take off the lids. Allow to cool in racks for at least 30. Rinse pots, blank slides racks and lids in water after use ALWAYS DO TWO FULL RACKS WHEN MICROWAVING SECTIONS; ESSENTIAL FOR STANDARDISATION ESPECIALLY IF VIA IS TO BE DONE Wash slides 2 x 5 in PBS Block for 30 at RT (use ~100 ul per slide), in a humid chamber. Blocking solution will vary depending on the 1 o Ab used.

7 Page: 7 of Tap off block but DO NOT wash in PBS. Wipe around section carefully and 1 o antibody (in block). The concentration of 1 o Ab will vary. Incubate in a humid chamber at 4 o C o/night. Day Remove the humid chamber from the fridge/cold room. Wash slides 2 x 5 in PBS Add 2 o antibody (will vary depending on antibody used). Incubate at RT for 60 in a humid chamber Wash slides 2 x 5 in PBS Add ABC or streptavidin reagent. Incubate at RT for 60 in a humid chamber Wash slides 2 x 5 in PBS In the fume hood, Drip DAB onto sections and leave until colour develops, this will vary depending on antibody used (have something white underneath to see colour more clearly Wash slides 2 x 5 in PBS Counterstain with weak Lillie-Mayer s haematoxylin ( 1 / 10 dilution of strong haematoxylin) for sec as needed Discard solution when counterstaining requires longer than 30 seconds and dilute and filter a fresh stock Can only use Lillie-Mayer s for VIA Dip several times in 4 changes of tap water Dehydrate and clear slides: x 5 100% EtOH, 3 x 5 xylene OR dips in 3 x 100% EtOH, dips in 3 x xylene Mount in mounting media as usual. Carefully squeeze out obvious air bubbles and allow slides to dry in the fume hood.

8 Page: 8 of 8 9. Maintenance: 9.1. Not Applicable 10. Shutdown / Disposal: DAB is to be disposed by being collected into an appropriately labelled receptacle and removed by an authorized chemical disposal contractor Xylene can be reused, so collect into an appropriately labelled bottle and store until needed. MUST NOT BE POURED DOWN SINK so therefore must be disposed of by an authorized chemical disposal contractor Small quantities of acetone and methanol can be allowed to evaporate in a fume hood Larger quantities of acetone and methanol should be collected into an appropriately labelled waste container and disposed by an authorised chemical disposal contractor Excess or expired quantities of hydrogen peroxide and mountant should be collected individually into appropriately labelled waste containers and disposed by an authorised chemical disposal contractor Lillie Mayer Haemotoxylin should be collected into an appropriately labelled waste container and disposed by an authorised chemical disposal contractor All other reagents can be poured down the sink with plenty of water Coplin Jars soak in 7X detergent and rinse in hot water, then soaked in 0.1M HCL, then rinse in ddh Change History: Issue Number: 2nd Issue Reviewed by: Dr Judith Weidenhofer Review Date: 6 May 2011 Date Issued: 26 October 2010