Take care to keep sections hydrated at all times. Drying out will lead to non-specific staining.
- De-paraffin and rehydrate slides
- Incubate 5’ each in xylenes (x2), then 3’ each in 100% EtOH (x2), 95% EtOH (x2) and 70% EtOH (x2)
- Put slide rack in PBS while preparing the next step
- Antigen retrieval (AR): water bath at 95oC-100oC for 25’ in 10 mM citrate buffer pH 6.0 or Tris-EDTA pH 9.0 (recommended)(10 mM Tris base + 1 mM EDTA and tween 0.05%, pH will be about 9 w/o adjustment). Pre-heat buffer in water bath. After 25’, allow slides to cool on benchtop at least 30’ then transfer to PBS 5’.
- Incubate sections with Image iT-signal enhancer (Invitrogen) (cover sections with several drops) for 30’ at RT (reduces background in green channel…prob. not necessary for red channel secondaries).
- Block in PBS + 1% BSA and 2% serum from the host of the secondary antibody (goat serum for Alexa-fluor secondaries; use 20 ml per ml)
- Segregate tissue sections from each other with a Pap Pen if necessary
- Add 100-250 ml of block to each tissue spot (depends on size, of course)
- Incubate at RT for 1 hour in a humidified chamber
- Bind tissues with primary antibodies
- Make up primary antibodies in PBS + 1% BSA at the appropriate dilutions, plan on 100-250 ml per section if using the pap pen.
- Dilute CK14 (LabVison, rabbit) to 1:500 and CK8/18 (Vector, mouse) to 1:500 in the PBS + 1% BSA.
- Make sure one section is stained with just the antibody diluent as a control.
- Incubate overnight at 4C in a humidified chamber, if desired, use probe clips for sections with larger area and/or only one section per slide