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A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. Xylene.
  2. Ethanol (anhydrous denatured, histological grade 100% and 95%).
  3. Hematoxylin (optional).
  4. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dHO, mix.
  5. Fixative Options: For optimal fixative, please refer to the product datasheet.
    1. 10% neutral buffered formalin.
    2. Acetone.
    3. Methanol.
    4. 3% formaldehyde: To prepare 100 ml, add 18.75 ml 16% formaldehyde to 81.25 ml 1X PBS.
  6. 10X Tris Buffered Saline (TBS) Wash Buffer: (#12498) To prepare 1 L 1X TBS add 100 ml of 10X TBS to 900 ml dHO, mix.
  7. Methanol/Peroxidase: To prepare, add 10 ml 30% HO to 90 ml methanol. Store at -20°C.
  8. Blocking Solution: 1X TBS/0.3% Triton™ X-100/5% Normal Goat Serum (#5425). To prepare, add 500 µl goat serum and 30 µl Triton™ X-100 to 9.5 ml 1X TBS.
  9. Detection System: SignalStain Boost IHC Detection Reagents (HRP, Mouse #8125; HRP, Rabbit #8114).
  10. Substrate: SignalStain DAB Substrate Kit (#8059).

B. Sectioning

  1. For tissue stored at -80°C: Remove from freezer and equilibrate at -20°C for approximately 15 min before attempting to section. This may prevent cracking of the block when sectioning.
  2. Section tissue at a range of 6–8 µm and place on positively charged slides.
  3. Allow sections to air dry on bench for a few min before fixing (this helps sections adhere to slides).