Note: take care to keep sections hydrated at all times.  Drying out will lead to non-specific staining.

  • De-paraffin and rehydrate slides
    1. Incubate 5’ each in xylenes (x2), then 3’ each in 100% EtOH (x2), 95% EtOH (x2) and 70% EtOH (x2)
    2. Put slide rack in PBS while preparing the next step
  • Antigen retrieval in 10 mM citrate buffer pH 6.0i
    1. Pre-heat buffer for at least 30 minutes before adding slides
    2. Incubate slides in buffer at 95oC-100oC for 25 minutes
    3. Let slides cool in AR buffer at RT for 30’
    4. Put slides in PBS while preparing the next step
      1. Our citrate buffer is made with citric acid (others are made with sodium citrate). Note you may also want to try Tris-EDTA pH 9.0 (10 mM Tris base + 1 mM EDTA and tween 0.05%) if you get poor staining with Citrate, this buffer may give higher background for some antigens but you can also often use the antibody at a more dilute concentration (and it has worked really well for some antibodies that did not work in citrate, such as GATA3)
  • Block endogenous peroxidases with H2O2
    1. Make up 3% H2O2 fresh (180 ml PBS + 20 ml 30% H2O2)
    2. Incubate for 10’
    3. Put slides in PBS while preparing the next step
  • Block in PBS + 1% BSA and 2% serum from the host of the secondary antibody (horse for anti-mouse, goat for anti-rabbit; use 20 ml per ml)
    1. Segregate tissue sections from each other with a Pap Pen if necessary
    2. Add 100-200 ml of block to each tissue spot (depending on size and use of pap pen)
    3. Incubate at RT for 1 hour in a humidified chamber
  • Bind tissues with primary antibodies
    1. Make up primary antibodies in PBS + 1% BSA at the appropriate dilutions, plan on 150 ml per section if using the pap pen.  If not using the pap pen or sections are large, scale up (this goes for all subsequent steps mentioning 150 µl)
    2. 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