Five-micron sections of embedded tissues were deparaffinized and rehydrated with xylene and ethanol. Slides were then blocked with blocking buffer (1% bovine serum albumin, 0.3% Triton X-100, in phosphate buffered saline (PBS)) for a minimum of 30 minutes. Monoclonal mouse anti-human HLA-G antibody 4H84 (Santa Cruz Biotechnology, Inc. Dallas, TX[24 (link)]) was applied at 4 °C for 8–12 hours. This antibody binds to both membrane-bound and soluble intracellular isoforms of HLA-G [25 (link)]. Slides were washed with PBS and then incubated with biotinylated rabbit-anti-mouse antibody (Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature. Signal was detected with VECTASTAIN R.T.U Elite ABC Reagent kit (Vector Laboratories) according to the manufacturer’s instructions. Slides were counter stained with the Brown-Hopps modification of the Gram stain (Fisher Scientific, St. Louis, MO) as previously described[21 (link)].
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Immunohistochemical Analysis of HLA-G Expression
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- Blocking with blocking buffer (1% bovine serum albumin, 0.3% Triton X-100, in phosphate buffered saline (PBS))
- Application of monoclonal mouse anti-human HLA-G antibody 4H84 at 4 °C for 8–12 hours
- Incubation with biotinylated rabbit-anti-mouse antibody for 30 minutes at room temperature
- Binding and detection of both membrane-bound and soluble intracellular isoforms of HLA-G
- Deparaffinization and rehydration of tissue sections with xylene and ethanol
- Washing slides with PBS
- Not explicitly mentioned
- Not explicitly mentioned
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