Drain and Dry
- The slides are coated with APES.
- After the sections are cut at 3µm, they are left to dry on the Red Carpet (from Osenses) at room temperature for 15 minutes.
- The slides are then placed in the oven at 60°C for 60 minutes.
- Please note the sections where cut 1 week earlier and kept in fridge (3-5C) prior to the staining. On the day of staining, the sections are removed from the fridge and placed on the bench to get to room temperature. The sections are then placed back into the oven at 60°C for 5 minutes to melt the wax again. This helps the dewaxing of the slides.
Dewaxing and rehydration of the tissues
- Placed the slides in xylene substitute 1 for 5 minutes
- Placed the slides in xylene substitute 2 for 3 minute
- Placed the slides in xylene substitute 3 for 3 minute
- Placed the slides in 100%EtOH 1 for 5 minutes
- Placed the slides in 100%EtOH 2 for 3 minute
- Placed the slides in 100%EtOH 3 for 3 minute
- Placed the slides in tap water for 5 minutes
Antigen retrieval (HIER) using PT Module - Used TRIS-EDTA pH 9.00
- Fill the right tank with 1.5L of Tris-EDTA and set the preheat temperature to 65°
- At 65°C the dewaxed slides are placed in the tank and cycle is started (20 minutes at 98°C). The ramp temperature from 65C to 98C is 15 min.
- Once the temperature is down to 85°C the tank is unlocked and the slide are placed immediately in the tap water for 5 minutes.
Staining of the slides
Done using the LabVision AutoStainer 360 and the Novocastra Novolink Polymer System and Candela Chromogen, using the protocol and regents listed below.
- The Peroxide Block used was in-house, 3% H2O2 prepared in MQ water
- The Protein block used was in-house prepared 0.2% LFDM in NaCl (containing 0.1% Tween-20) then filtered thru 0.2 µm.
- Wash buffer is NaCl-T pH 7 (final working concentration: 200 mM NaCl; 0.1% Tweeen-20) prepared with RO water. The working NaCl-T buffer should be prepared freshly and should appear translucent.
- The Novolink Polymer and Osenses' Candela Chromogen (30µl/1 ml reaction buffer)
- The run of 36 slides takes 2 hour and 50 minutes to finish, 6.0 litres of TBST Wash. Buffer and 6 litres of water. But to make sure, prepare 7.0 L TBST for 36 slides.
Wash Buffer->Peroxide Block (5 min)->Wash Buffer (2x)->Protein Block (5 min)->Wash Buffer (2x)->Primary antibody (30 min)->Wash Buffer (2x)->Novolink Polymer (30 min)->Wash Buffer (3x)->Water wash (1x)
- Osenses' Candela chromogen (30 µl chromogen is added to 1 ml reaction bufffer).
- The resulting solution was applied to each slide for 5 minutes.
- The slides where then washed well in Tap Water.
- The slides are placed into a sliver rack and placed in filtered Haematoxylin (from Leica) for 30 seconds. The slides are then washed well in Tap Water.
Cover slipping the slides
- Placed the slides in 100%EtOH 1 for 2 minutes
- Placed the slides in 100%EtOH 2 for 1 minute
- Placed the slides in 100%EtOH 3 for 1 minute
- Place the slides in xylene substitute 1 for 1 minute
- Place the slides in xylene substitute 2 for 1 minute
- Place the slides in xylene substitute 3 for 1 minute
- Picked up one slide at a time and place one drop of DPX mounting medium on the coverslip and place the slide on top. Avoid air bubbles.
- The slides where then allowed to dry
Cleaning the Autostainer
- At the end of each day – pump 10% bleach though the water and buffer lines. Leave this for 10 minutes and then pump RO water though both lines.
- Every 240 slides – the Autostainer will ask you do clean the probe.
- Mix 5ml of 11.94M HCL with 95ml of ethanol, pour the mixture into the sink and wait 30 minutes.
- Check for at least 3.5 litres of water
- In a vial mix 0.5ml of DAB-Away reagent 1, 0.5 ml of DAB-Away reagent 2 and 4.0ml of MQ water. Put this vial in position A4 (left rear corner).
- Put 5.0ml of DAB Decolourizer in vial B4.
- Click the OK button.