IHC-P Immunohistochemistry – Parafin staining

 

  1. Tissue Sectioning
  • Make 4-µm sections and place on pre-cleaned and charged microscope slides
  • Heat in a tissue-drying oven for 45 min at 60°C
  1. Deparaffinization
  • Wash slides in 3 changes of xylene – 5 min each at RT
  1. Rehydration
  • Wash slides in 3 changes of 100% alcohol – 3 min each at RT
  • Wash slides in 2 changes of 95% alcohol – 3 min each at RT
  • Wash slides in 1 change of 80% alcohol – 3 min at RT
  • Rinse slides in gentle running distilled water – 5 min at RT
  1. Antigen retrieval
  • Heat the slides in 0.01 M sodium citrate buffer, pH 6.0 at 120°C (pressure pan) – 2 min
  • Remove from heat and let stand at RT in buffer – 20 min
  • Rinse in 1X TBS with Tween (TBST) – 1 min at RTR
  1. Immunostaining
  • Do not allow tissues to dry at any time during the staining procedure
  • Apply a universal protein block – 1h at RT
  • Drain protein block from slides, apply diluted primary antibody (5-10 µg/ml) (1:50-1:200) – overnight at 4°C
  • Rinse slides in 1X TBST – 1 min at RT
  • Apply a biotinylated secondary antibody (specific to the host of the primary antibody) – 1h at RT
  • Rinse slides 1X TBST – 1 min at RT
  • Apply alkaline phosphatase streptavidin – 30 min at RT
  • Rinse slides in 1X TBST – 1 min at RT
  • Apply alkaline phosphatase chromogen substrate – 30 minutes at RT
  • Wash slides in distilled water – 1 minute at room temperature.
  1. Dehydrate
  • This method should only be used if the chromogen substrate is alcohol insoluble
  • Wash slides in 2 changes of 80% alcohol – 1 min each at RT
  • Wash slides in 2 changes of 95% alcohol – 1 min each at RT
  • Wash slides in 3 changes of 100% alcohol – 1 min each at RT
  • Wash slides in 3 changes of xylene – 1 min each at RT
  • Apply coverslip