AIF subcellular localization analysis and determination of AIF pathology score

1. Immunofluorescence Protocol

• Wash slides with 100% Xylene (5 min)
  
  Antigen Retrieval Solution
  9.9mL DiH₂O
  100µL of 1M Tris HCI
  10µL Proteinase K
   
• Wash slides with 100% Xylene (5 min)
• Wash slides with 100% Xylene (5 min)
• Wash slides with 100% Ethanol (5 min)
• Wash slides with 75% Ethanol (5 min)
• Wash slides with 30% Ethanol (5 min)
• Wash slides with 1X PBS (5 min)
• Wash slides with 1X PBS (5 min)
• Add 250 µL of the Antigen Retrieval Solution directly to slides AFTER moving slides to the incubator (this ensures solution doesn’t fall off during transport) at 37°C (30 min) and don’t forget to place wet papers in the petri dish and cover top back on (this keeps the chambers from drying out).
• Wash slides with 1X PBS (5 min)
• Wash slides with 1X PBS (5 min)
• Block the slides with 5% BSA and 0.1% Triton X-100 in 1X PBS (Blocking Solution) for 1 hour at room temperature.  The blocking solution needs to be filtered at 0.45 µm filter and don’t forget to place wet papers in the petri dish and cover.
  
  Blocking Solution
  25mL 1X PBS
  1.25g BSA
  25µL Triton X
   
• Wash slides with 1X PBS (5 min)
• Wash slides with 1X PBS (5 min)
• Incubate slides with primary antibody cocktail according to manufacturer's suggested concentration prepared with Blocking Solution. (Hao Ding: This is where your protocol may defer depending on which antibody you are selecting from which company, vendor, etc).  Add 250 µL of the primary antibody while the slides are in the refrigerator (overnight at 4°C), in their chambers.
• Wash slides with 1X PBS (5 min)
• Wash slides with 1X PBS (5 min)
• Incubate slides with secondary antibody cocktail (250 µL) prepared with Blocking Solution at room temperature (1 hour) with wet papers in the petri dish and cover. (Hao Ding: This is where your protocol may defer depending on which secondary antibodies  you are selecting from which company, vendor, etc).   
• Place tinfoil over the petri dishes to prevent light exposure.
• Wash slides with 1X PBS (5 min)
• Wash slides with 1X PBS (5 min)
• Dry the slides with Kim wipes and avoid touching the sample.
• Add one drop of the ProLong DAPI Antifade solution to each slide directly in the middle of the slide. Place the cover slip on top of the slide and allow Prolong DAPI Antifade solution to spread on its own.  Use blunt tweezers to gently press on coverslip to move any bubbles to edges.
• Leave the slides at room temperature (overnight, covered).
• The following day, trace the edges of the coverslip with clear fingernail polish.

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